KR102133931B1 - Acetaminophen responsive genes in hydra magnipapillata and the method for diagnosing aquatic environment pollution using the same - Google Patents

Abstract

The present invention relates to acetaminophen responsive genes in Hydra magnipapillata and an aquatic environment pollution diagnosis method using the same. As a result of analyzing genes changed after exposing Hydra magnipapillata to acetaminophen, it is proved that an expression ratio is changed in 32 kinds of genes from a group exposed to acetaminophen of 5,000 microgram/L for 12 hours, in 66 kinds of genes from a group exposed to acetaminophen of 5,000 microgram/L for 24 hours, in 37 kinds of genes from a group exposed to the same for 48 hours, and in 84 kinds of genes from a group exposed to the same for 70 hours, 25 kinds of genes expected to be related to physiology and metabolism in a live cell are screened from differentially expressed genes, and it is expected that exposure to acetaminophen affects which biological functions of Hydra magnipapillata through function analysis of the screened genes. Accordingly, 25 kinds of genes can be usefully used as a biomarker capable of checking exposure to acetaminophen.

Description

{Acetaminophen responsive genes in hydra magnipapillata and the method for diagnosing aquatic environment pollution using the same}

The present invention relates to a hydra gene corresponding to acetaminophen exposure and a method for diagnosing environmental pollution in aquatic ecosystem using the same.

For the treatment of diseases, medicines are very important necessities in modern life. As a result of analyzing the water quality of sewage treatment plants in cities across the country, cholesterol lowering agents, anti-inflammatory and antipyretic analgesics due to indispensable drugs that were indiscriminately discarded were detected. These medicines are widely used not only in humans, but also in animal and agricultural products production and aquaculture, and after being administered to humans or animals, some of them are converted into metabolites as they are or in vivo, excreted in urine or feces and enter the environment. In addition, medicines that are not used or used at home are discarded in the trash cans and toilets, and medicines that have been produced by pharmaceutical companies and have exceeded their expiration date should be discarded and discarded in the environment as they are, which may affect the health of people due to environmental pollution such as water quality. Or it will disturb the ecosystem. Recently, medicines that remain in the environment are being considered as new pollutants, and several studies have been published to increase the pollution of rivers and soils due to the continuous release due to use and disposal.

Acetaminophen is a typical antipyretic analgesic ingredient and is used to relieve fever, headache, muscle pain, and toothache. Acetaminophen relieves pain by inhibiting the activity of cyclooxygenase-2. According to a 2003 study, over 400 tonnes of acetaminophen was consumed in the UK in 2000 (Sebastine and Wakeman, 2003, Process Saf. Environ. 81: 229-235). In the environment, it was detected up to 12.24-200 μg/L (Villar-Navarro et al., 2018, Water Research, 139: 19-29; Gheorghe et al., 2016, Environ. Monit. Assess. 188: 379; Togola and Budzinski, 2008 , J. Chromatogr. A. 1177: 150-158). Due to the growth rate of the population and the increase in the source of stress, its use is thought to increase every year. Therefore, in order to diagnose the pollution of aquatic environment by medicines and to predict their biological risk, risk studies of aquatic animal models are needed.

On the other hand, Hydra magnipalillata is the first multicellular animal with neurons and occupies a very important position in the study of biological evolution. As a diploid animal, reproduction is by asexual and sexual reproduction, and has excellent regeneration ability. Morphology-related signaling systems (Hobmayer et al., 2000, Nature 407: 186-189; Arvizu et al., 2006, Differentiation 74: 305-312; Augustin et al., 2006, Dev. Biol. 296: 62-70; Kaesbauer et al., 2007 , Bev. Biol. 303: 376-390), Signals and signal transduction systems associated with regeneration (Bode, 2003, Dev. Dyn. 226: 225-236; Fujisawa, 2003, Dev. Dyn. 226: 182-189; Holstein et al. , 2003, Dev. Dyn. 226: 257-267), studies related to signaling of cell differentiation (Thomsen et al., 2004, Mech. Dev. 121: 195-204; Philipp et al., 2005, Gene Expr. Patterns, 5: 397-402) and tissue fate determination signals (Bielen et al., 2007, Development, 134: 4187-4197). These signaling systems are common to all animals through the evolutionary process, so it is possible to extrapolate the abnormality of the signaling system in the hydra to other animals. In most animals, various genes that are expressed only during the early stages of development are maintained in adults in hydra. Therefore, it is possible to study the signal transduction system related to it without any time limitation, and molecular markers for each cell type are known, so it is possible to study cell differentiation. In addition, in situ hybridization (ISH) and immunohistochemistry (IHC) methods for localization of gene and protein expression have been established, and ESTs and genomic data of 160,000 clones are now database (DB). (Chapman et al., 2010, Nature 464: 592-596), so it is possible to search for information on genes belonging to various signaling systems. The importance of hydra as an alternative laboratory animal for environmental risk studies has also been discussed (Yum et al., 2014, Mol. Cell. Toxicol. 10: 339-346).

Accordingly, the present inventors extracted 17,000 singletons from gene information accumulated in the DDBJ/EMBL/NCBI gene database to secure and detect specific gene candidates for acetaminophen exposure. The oligo-probe was designed for the extracted singletons, and a 17K Hydra Express Gene Microarray (HEGEM) was loaded with them. By discovering a gene whose expression level is changed by exposure to acetaminophen using the 17K HEGEM, a biomarker capable of confirming exposure to acetaminophen, a method of confirming exposure using it, and physiology of hydra by exposure to acetaminophen The present invention was completed by establishing a method for predicting metabolic changes.

An object of the present invention to provide a hydra (Hydra magnipapillata) gene, and if acetaminophen exposure using the same biological function or change prediction method corresponding to acetaminophen exposure.

In order to achieve the above object, the present invention is a microarray chip for detecting the presence of acetaminophen (Acetaminophen), in which all oligonucleotides of each nucleic acid sequence of SEQ ID NOs: 1 to 25, or complementary strand molecules thereof, are integrated. microarray chip).

In addition, the present invention comprises the steps of: 1) separating RNA from the hydra of the experimental group exposed to the test sample and the hydra of the normal control, respectively; 2) synthesizing cDNA from RNA of the experimental group and the control group of step 1) and labeling the experimental group and the control group with different fluorescent materials; 3) hybridizing the cDNA labeled with different fluorescent materials of step 2) with the microarray chip of claim 1; 4) analyzing the reacted microarray chip; And 5) confirming the degree of gene expression integrated in the microarray chip of claim 1 in comparison with the control group in the analyzed data.

In addition, the present invention comprises the steps of: 1) separating RNA from the hydra of the experimental group exposed to the test sample and the hydra of the normal control, respectively; 2) Quantitative real-time reverse transcript polymerase chain reaction using the RNA of step 1) using both primer pairs complementary to each gene of SEQ ID NOs: 1 to 25 and capable of amplifying the gene. , qRT-PCR); And 3) comparing the gene product of step 2) with the control to confirm the expression level, and provides a method for detecting whether acetaminophen is exposed in the sample.

In addition, the present invention provides a kit for detecting whether acetaminophen is exposed, including the microarray chip.

In addition, the present invention provides a kit for detecting whether or not acetaminophen is exposed, including all primer pairs complementary to each gene described in SEQ ID NOs: 1 to 25 and capable of amplifying the gene.

The present invention analyzed the hydra gene corresponding to the exposure of acetaminophen, and exposed to 5000 µg/L of acetaminophen 32 types in a 12 hour exposure group, 66 types in a 24 hour exposure group, 37 types in a 48 hour exposure group, and 72 hours exposure. In the group, 84 genes were confirmed to be changed in expression, and among the differentially expressed genes, 25 genes expected to be related to physiology and metabolism in vivo were selected, and acetaminophen was analyzed through functional analysis of the selected genes. It can be predicted which biological function of the exposure of Hydra, the 25 genes can be useful as a biomarker to confirm the exposure to acetaminophen.

Hereinafter, the present invention will be described in detail.

The present invention has discovered a gene derived from Hydra magnipapillata whose expression changes in response to exposure to acetaminophen, and a microarray chip incorporating a hydra-derived gene whose expression level changes with respect to the exposure to acetaminophen is acetaminophen. Can be used to detect exposure and diagnose aquatic contamination.

The present invention provides a microarray chip for detecting the presence or absence of exposure to acetaminophen, in which all of the oligonucleotides of each nucleic acid sequence of SEQ ID NOs: 1 to 25, or complementary strand molecules thereof, are integrated.

The gene may be derived from Hydra magnipapillata .

The microarray chip for detecting the presence or absence of acetaminophen may be produced by a method known to those skilled in the art. Specifically, the searched gene can be immobilized on a substrate of a microarray chip using a probe. At this time, a micropipetting method using a piezoelectric method or a method using a pin type spotter may be used. The substrate of the microarray chip may be coated with at least one active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be manufactured to be selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose film.

In addition, the present invention comprises the steps of: 1) separating RNA from the hydra of the experimental group exposed to the test sample and the hydra of the normal control, respectively; 2) synthesizing cRNA labeled with different fluorescent substances from the RNA of the experimental group and the control of step 1); 3) hybridizing the cRNA labeled with the different fluorescent materials of step 2) with the microarray chip of claim 1; 4) analyzing the reacted microarray chip; And 5) confirming the degree of gene expression integrated in the microarray chip of claim 1 in comparison with the control group in the analyzed data.

The sample may be any one selected from the group consisting of biological samples, food samples, chemical samples, industrial samples, clinical samples and environmental samples.

The fluorescent material of step 2) may be any one selected from the group consisting of Cy3, Cy5, FITC (poly L-lysine-fluorescein isothiocyanate), RITC (rhodamine-B-isothiocyanate) and rhodamine.

In addition, the present invention comprises the steps of: 1) separating RNA from the hydra of the experimental group exposed to the test sample and the hydra of the normal control, respectively; 2) Quantitative real-time reverse transcript polymerase chain reaction using the RNA of step 1) using both primer pairs complementary to each gene of SEQ ID NOs: 1 to 25 and capable of amplifying the gene. , qRT-PCR); And 3) comparing the gene product of step 2) with the control to confirm the expression level, and provides a method for detecting whether acetaminophen is exposed in the sample.

The sample may be any one selected from the group consisting of biological samples, food samples, chemical samples, industrial samples, clinical samples and environmental samples.

In addition, the present invention provides a kit for detecting whether acetaminophen is exposed, including the microarray chip.

The kit may additionally include any one selected from the group of fluorescent materials consisting of a strepavidin-like phosphatease conjugate, a chemiflurorensce, and a chemiluminescent material. .

In addition, the kit is a buffer solution used for hybridization, reverse transcriptase for synthesizing cDNA (complementary DNA) from RNA, deNucleotide triphosphates (dNTPs) and rNTPs (ribonucleotide triPhosphates, pre-mixed or separated supply type), labeling reagent, and washing It may further include any one selected from the group of reaction reagents consisting of a buffer solution.

In addition, the present invention provides a kit for detecting whether acetaminophen is exposed, including all primer pairs complementary to each gene described in SEQ ID NOs: 1 to 25 and capable of amplifying the gene.

In a specific embodiment of the present invention, the present inventors determined the tofu and tentacle regeneration anti-inhibitory concentrations for acetaminophen (Inhibitory concentration 50, IC ₅₀ ) in order to discover a gene derived from hydra that is changed by exposure to acetaminophen and determine 5000 After culturing by exposing 20 individuals of Hydra to µg/L for 12, 24, 48, and 72 hours, RNA was isolated, and genes that were changed more than 2 times compared to the control were selected. As a result, 32 types in the 12-hour exposure group (31 increase, 1 decrease; Table 2 and Table 3), 66 in the 24-hour exposure group (61 increase, 5 decreases; Table 4 and Table 5), 48 In the time-exposed group, 37 genes (33 increased, 4 decreased; Table 6 and Table 7), and in the 72-hour exposed group, the gene expression was doubled in 84 (81 increased, 3 reduced; Table 8 and Table 9). It was confirmed that the abnormal change (see Table 2 to Table 9). In addition, among these differentially expressed genes, 25 genes expected to be related to physiology and metabolism in vivo were selected (see Table 10).

Therefore, through analysis of the functions of the genes, it is possible to predict which biological function of exposure to acetaminophen affects hydra, and the 25 genes can be usefully used as a biomarker to confirm exposure to acetaminophen.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only for illustrating the present invention, and the present invention is not limited by the following examples.

<Example 1> Preparation of hydra microarray

For the production of a hydra microarray , we designed an oligo-probe based on the 17,000 singlele gene information from Hydra magnipapillata accumulated in the DDBJ/EMBL/NCBI gene database. Synthesis and mounting on a slide glass produced a 17K Hydra Express Gene Microarray (HEGEM).

<Example 2> Separation of genes from hydra exposed to acetaminophen

<Example 2-1> Hydra culture

Hydra magnipapillata wild strain 105 is pre-sold by the National Institute of Genetics, Yata, Mishima, Japan, and cultured (1 mM NaCl, 1 mM CaCl ₂ , 0.1 mM KCl, 0.1 mM MgSO ₄ , 1 mM Tris (hydroxymethyl) aminoethane (pH 7.6)). The water temperature was fixed at 18°C, and Artemia larvae hatched freshly once every two days were fed as food. After feeding, the culture was changed a few hours later.

<Example 2-2> Establishing exposure conditions of acetaminophen

In order to establish the acetaminophen exposure conditions for hydra, the concentration of tofu and tentacle regeneration for acetaminophen (Inhibitory concentration 50, IC ₅₀ ) was determined (Table 1).

The exposure concentrations for differential expression gene detection were exposed to 5,000 µg/L corresponding to about 1/50 of the IC ₅₀ (231.3 mg/L) at 72 hours for 20, 12, 24, 48 and 72 hours, respectively.

Table 1 shows the acetaminophen half inhibition concentration in hydra.

Exposure time IC ₅₀ (unit mg/L) 72 hours 231.3 96 hours 487.9

<Example 2-3> RNA extraction and purification

Tri-Reagent solution (Molecular Research) in the hydra tissue of each exposed experimental group and unexposed control group of 12, 24, 48 or 72 hours exposed to acetaminophen (5000 µg/L) according to the conditions of <Example 2-2> Center Inc, Cincinnati, Ohaio, USA) 1 ml was added, homogenized using a glass homogenizer and left at room temperature for 10 minutes. After 200 µl of chloroform was added and mixed well, the mixture was left at room temperature for 10 minutes, and centrifuged (12,000 x g, 4°C) for 15 minutes. Then, a supernatant was obtained, 500 μl of isopropanol was added, and the mixture was left at room temperature for 5 minutes. After centrifugation for about 20 minutes (12,000×g, 4°C), the solution was removed to collect only the precipitate. 50 μl of 70% ethanol solution was added to the precipitate, followed by centrifugation for 5 minutes, and then the ethanol solution was removed and precipitated. RNA was dried. After drying, it was dissolved in sterile water treated with an appropriate amount of diethylpyrocarbonate (DEPC).

<Example 2-4> fluorescent substance labeled cRNA synthesis

The total RNA (total RNA) of the exposure experiment group and the control purified in <Example 2-3> was Cy3 and as follows according to the manufacturer's protocol using Agilent's Low RNA Input Linear Amplification Kit Plus (Agilent Technologies, USA). It was labeled with Cy5.

Specifically, 1 μg of RNA was mixed with dT-T7 promoter primer (dT-promoter primer) and MMLV-Reverse transcriptase to perform a reverse transcription reaction at 40° C. for 2 hours. Then, T7 polymerase was added to perform linear amplification at 40° C. for 2 hours. Through the amplification process, samples of the experimental group and the control group were labeled with Cy3-CTP and Cy5-CTP, respectively.

<Example 3> microarray hybridization and analysis

<Example 3-1> Microarray hybridization

In <Example 2-4>, the fluorescently labeled cRNA sample was purified using a Qiagen PCR purification kit, and eluted with distilled water. After adding the purified fluorescent label-cRNA sample to the hybridization buffer (3×SSC, 0.3% SDS, 50% formamide, 20 μg Cot-1 DNA, 20 μg yeast tRNA) , concentrated with microcon YM-30 to make a hybridization mixture. The hybridization mixture was denatured by heating to 95° C. for 3 minutes, and centrifuged at 12,000×g for 30 seconds to lower the temperature.

A cover slip was covered on the hydra microarray prepared in <Example 1>, and the hybridization mixture was pipetted. Thereafter, the microarray was placed in a GT-Hyb chamber (Chamber) and reacted at 65° C. for 16 hours to hybridize, and then the microarray was removed from the chamber, washed, rotated to dry the microarray, and then scanned in a dark room until scanning. Kept. The experimentally completed hydra oligo-microarray was scanned using the Axon GenePix 4000B scanner (Axon Instrument, USA), and each point from the scanned image is gridded using a gridding file using the GenePix Pro 6.0 program. Then, it was quantified to obtain a GPR file (GPR file) containing analysis values such as Cy5/Cy3 intensity and ratio of each point.

<Example 3-2> Analysis of microarray

From the GPR file obtained in the GenePix Pro Program, the analysis was performed as follows using the analysis program GeneSpring 7.3.1 (Agilent Technologies, USA).

Normalization was performed using LOWESS (locally weighted regression scatterplot smoothing), and a reliable gene has a median sum lower than the background or a standard deviation of each pixel value. Significant genes were obtained by flagging out points that were not significant. Significant genes were selected for those with a normalized ratio value of more than twice the difference.

<Example 3-3> Confirmation of specific gene candidate group

Firstly, genes in which the amount of expression of acetaminophen was changed by 2 times or more according to the exposure time were selected.

As a result, 32 types in the 12-hour exposure group (31 increase, 1 decrease; Table 2), 66 in the 24-hour exposure group (61 increase, 5 decreases; Table 3), 37 in the 48-hour exposure group (33 types of increase, 4 types of decrease; Table 4), it was confirmed that the gene expression of 84 types (81 types of increase, 3 types of decrease; Table 5) in the 72-hour exposure group was changed more than 2 times (Table 2 to Table 5). Reference).

Genes with increased expression in acetaminophen (5,000 μg/L) 12-hour exposure group (31 types) GenBank Acc. Gene name Expression change XM_002159705 tetratricopeptiderepeatdomain18 5.5 XM_002163272 Calmodulin (CaM) 5 XM_002166284 nucleartranscriptionfactorY,gamma 5 XM_002171100 LINE1-likeRNA-directed DNA polymerase 4.7 XM_002163542 thrombospondintype1repeat-containing protein2 4.1 XM_002165417 Aldehydedehydrogenasetype III 4.1 XM_002168891 hypotheticalproteinLOC100200621 4 XM_002155406 hypotheticalproteinLOC100209558 3.9 XM_002164217 transcriptionfactorIIIA,putative 3.8 XM_002166497 predicted protein 3.8 XM_002171093 ZincfingerBEDdomain-containingprotein1 3.7 XM_002167903 transposase(putative) 3.5 XM_002156219 Y64G10A.7 (LOC100199089) 3.5 XM_002166728 predicted protein (LOC100202743) 3.3 XM_002166002 AGAP001049-PA (LOC100203666) 3.3 XM_002162428 predicted protein (LOC100206761) 3 XM_002154102 conserved hypothetical protein (LOC100207416) 3 XM_002166349 vasa-relatedproteinCnVAS1 2.8 XM_002159819 hypothetical protein LOC100200934 (LOC100200934) 2.8 XM_002163019 Y52D5A.1 (LOC100204794) 2.7 XM_002155169 PIF/Harbinger-likeprotein 2.7 XM_002169690 RCC2homolog 2.6 XM_002159385 gamete fusion-like protein (FusM) 2.4 XM_002158646 smallheatshockprotein 2.4 XM_002168296 predicted protein (LOC100206775) 2.3 XM_002154887 predicted protein (LOC100204881) 2.3 XM_002166161 mucin2 2.3 XM_002154859 conserved hypothetical protein (LOC100211478) 2.2 XM_002162284 Adalprotein 2.2 XM_002169727 transposasedomain-containingprotein 2.1 XM_002155419 hypotheticalproteinLOC100202204 2

Acetaminophen (5,000 μg/L) gene with reduced expression in the 12-hour exposure group (1 type) GenBank Acc. Gene name Expression change XM_002155491 oxidativestressprotein -2.5

Genes with increased expression in acetaminophen (5,000 μg/L) 24-hour exposure group (61 types) GenBank Acc. Gene name Expression change XM_002169214 predicted protein (LOC100211057) 8.5 XM_002168296 predicted protein (LOC100206775) 7.4 XM_002159819 hypotheticalproteinLOC100200934 7.2 XM_002159705 tetratricopeptiderepeatdomain18 6.6 XM_002160201 conserved hypothetical protein
(LOC100208305) 6.4 XM_002156369 MGC85118protein 6.2 XM_002168564 predicted protein (LOC100204874) 5.7 XM_002162820 EGF-like-domain,multiple3 5.6 XM_002156707 tyrosinekinasereceptor 4.7 XM_002164217 transcriptionfactorIIIA,putative 4.6 XM_002156219 Y64G10A.7 (LOC100199089) 4.4 XM_002155090 periculin 4.3 XM_002154916 hypothetical protein LOC100213187 4.2 XM_002154329 ATP-dependentDNAhelicasePIF1 4.2 XM_002168496 queuinetRNAribosyltransferase,putative 4.1 XM_002166002 AGAP001049-PA (LOC100203666) 4 XM_002171100 LINE1-likeRNA-directed DNA polymerase 3.9 XM_002158742 hypotheticalproteinLOC100200450 3.8 XM_002163648 hypotheticalproteinLOC100198744 3.8 XM_002166181 periculin 3.7 XM_002155169 PIF/Harbinger-likeprotein 3.5 XM_002170619 hepatopancreaskazal-typeproteinaseinhibitor 3.5 XM_002159692 predicted protein (LOC100207915) 3.4 XM_002162162 predicted protein (LOC100210397) 3.3 XM_002165249 ViralA-typeinclusionproteinrepeat,putative 3.3 XM_002165146 TPR 3.1 XM_002154685 predicted protein (LOC100210731) 3.1 XM_002158049 GSTM2protein 3.1 XM_002164282 embryonic-1 3.1 XM_002167903 transposase(putative) 3 XM_002163906 F59H6.5 (LOC100200858) 3 XM_002163049 mucin1,cellsurfaceassociated 2.8 XM_002159360 predicted protein (LOC100212284) 2.8 XM_002158818 hypothetical protein LOC100213161 2.8 XM_002155086 fascin2 2.8 XM_002155187 AGAP001049-PA (LOC100204965) 2.7 XM_002169654 predicted protein (LOC100206796) 2.7 XM_002168770 predicted protein (LOC100212392) 2.6 XM_002170755 Os02g0236500 (LOC100202302) 2.5 XM_002163554 hypotheticalproteinLOC100212404 2.4 XM_002158978 predicted protein (LOC100208242) 2.4 XM_002169621 SCO1-like,putative 2.3 XM_002170132 PMS1proteinhomolog1 2.3 XM_002166009 conserved hypothetical protein
(LOC100203206) 2.3 XM_002171093 ZincfingerBEDdomain-containingprotein1 2.3 XM_002163019 Y52D5A.1 (LOC100204794) 2.3 XM_002161961 predicted protein (LOC100205381) 2.3 XM_002162252 putativeascorbateperoxidase 2.2 XM_002158646 smallheatshockprotein 2.2 XM_002167113 predicted protein (LOC100202830) 2.2 XM_002154102 conserved hypothetical protein
(LOC100207416) 2.2 XM_002165417 Aldehydedehydrogenasetype III 2.2 XM_002166497 predicted protein (LOC100210282) 2.2 XM_002171073 hypothetical protein LOC100205096 2.2 XM_002154859 conserved hypothetical protein
(LOC100211478) 2.2 XM_002157841 DAZ-likeprotein 2.2 XM_002169968 embryonic-1 2.2 XM_002159385 gamete fusion-like protein (FusM) 2.1 XM_002167229 putative3-glucanase 2.1 XM_002169727 transposasedomain-containingprotein 2.1 XM_002165378 predicted protein (LOC100205682) 2.1

Acetaminophen (5,000 μg/L) gene with reduced expression in the 24-hour exposed group (5 types) GenBank Acc. Gene name Expression change XM_002154713 verylargeinducibleGTPase1 -2 XM_002166335 tyrosinekinasereceptor -2.2 XM_002155491 oxidativestressprotein -2.8 XM_002169302 hypotheticalproteinLOC100206448 -9.6 XM_002169831 hypotheticalproteinLOC100209805 -11.2

Acetaminophen (5,000 μg/L) gene with increased expression in the 48-hour exposure group (33 types) GenBank Acc. Gene name Expression change XM_002160201 conserved hypothetical protein
(LOC100208305) 8.4 XM_002167113 predicted protein (LOC100202830) 5.5 XM_002167592 H28G03.4 (LOC100214969) 5.1 XM_002164217 transcriptionfactorIIIA,putative 4.4 XM_002168496 queuinetRNAribosyltransferase,putative 4.1 XM_002156219 Y64G10A.7 (LOC100199089) 3.8 XM_002156707 tyrosinekinasereceptor 3.7 XM_002163801 immediateearlyproteinhomolog-related 3.7 XM_002159143 ATP-bindingcassette,sub-familyB(MDR/TAP),member6 3.6 XM_002169812 Os02g0236500 (LOC100204594) 3.6 XM_002168891 hypothetical protein LOC100200621 3.4 XM_002167288 hypothetical protein LOC100214587 3.4 XM_002154329 ATP-dependent DNA helicase PIF1 3.3 XM_002163019 Y52D5A.1 (LOC100204794) 3 XM_002159819 hypothetical protein LOC100200934 2.9 XM_002166002 AGAP001049-PA (LOC100203666) 2.9 XM_002169621 SCO1-like,putative 2.7 XM_002171093 ZincfingerBEDdomain-containingprotein1 2.7 XM_002171100 LINE1-likeRNA-directed DNA polymerase 2.7 XM_002159385 gamete fusion-like protein (FusM) 2.6 XM_002170630 Uncharacterized protein F54H12.3 (LOC100207145) 2.6 XM_002154887 predicted protein (LOC100204881) 2.6 XM_002167903 transposase(putative) 2.5 XM_002154102 conserved hypothetical protein (LOC100207416) 2.4 XM_002155187 AGAP001049-PA (LOC100204965) 2.4 XM_002155169 PIF/Harbinger-like protein 2.3 XM_002168770 predicted protein (LOC100212392) 2.3 XM_002157518 predicted protein (LOC100212520) 2.2 XM_002170755 Os02g0236500 (LOC100202302) 2.1 XM_002166349 vasa-relatedproteinCnVAS1 2.1 XM_002158934 predicted protein (LOC100201066) 2.1 XM_002168861 predicted protein (LOC100203601) 2 XM_002166728 predicted protein (LOC100202743) 2

Acetaminophen (5,000 μg/L) gene with reduced expression in the 48-hour exposure group (4 types) GenBank Acc. Gene name Expression change XM_002160019 Y20F4.5 (LOC100212801) -2.1 XM_002155491 oxidativestressprotein -2.6 XM_002169302 hypotheticalproteinLOC100206448 -2.7 XM_002169831 hypotheticalproteinLOC100209805 -2.8

Acetaminophen (5,000 μg/L) gene with increased expression in the 72-hour exposure group (81 species) GenBank Acc. Gene name Expression change XM_002169214 predicted protein (LOC100211057) 483.7 XM_002164282 embryonic-1 93.7 XM_002155090 periculin 55.2 XM_002154916 hypothetical protein LOC100213187 53.6 XM_002158742 hypothetical protein LOC100200450 48 XM_002169968 embryonic-1 42.7 XM_002166181 periculin 42.6 XM_002153775 hypothetical protein LOC100206472 38 XM_002158049 GSTM2 protein 33.6 XM_002158818 hypothetical protein LOC100213161 21.9 XM_002159692 predicted protein (LOC100207915) 19.5 XM_002170619 hepatopancreas kazal-type proteinase inhibitor 18.1 XM_002162252 putative ascorbate peroxidase 15.1 XM_002170829 GI18027 (LOC100199297) 12.2 XM_002160201 conserved hypothetical protein (LOC100208305) 10.9 XM_002167229 putative 3-glucanase 10.7 XM_002155086 fascin 2 10.3 XM_002159040 type II transmembrane C-type lectin 10 XM_002159617 CDK5 regulatory subunit associated protein 1 9.6 XM_002157290 conserved hypothetical protein (LOC100205450) 7.8 XM_002171073 hypothetical protein LOC100205096 6.4 XM_002156707 tyrosine kinase receptor 6.1 XM_002154033 Hydroxymethylglutaryl-CoA lyase, mitochondrial 5.9 XM_002167427 T-box protein AmphiTbx6/16 5.3 XM_002165146 TPR 5.2 XM_002163019 Y52D5A.1 (LOC100204794) 5.2 XM_002163801 immediate early protein homolog-related 5.1 XM_002161879 chromosome 21 open reading frame 29 5 XM_002155917 predicted protein (LOC100204912) 4.9 XM_002166002 AGAP001049-PA (LOC100203666) 4.7 XM_002167592 H28G03.4 (LOC100214969) 4.6 XM_002166497 predicted protein (LOC100210282) 4.6 XM_002154329 ATP-dependent DNA helicase PIF1 4.4 XM_002166161 mucin 2 4.4 XM_002163272 Calmodulin (CaM) 4.3 XM_002168496 queuine tRNA ribosyltransferase, putative 4.2 XM_002156219 Y64G10A.7 (LOC100199089) 4.2 XM_002168564 predicted protein (LOC100204874) 4.1 XM_002164217 transcription factor IIIA, putative 4.1 XM_002156622 tyrosine kinase receptor 4 XM_002167113 predicted protein (LOC100202830) 3.9 XM_002166889 putative endo-1,3-beta-glucanase 3.9 XM_002161814 Cnnos1 protein (Cnnos1) 3.7 XM_002161183 chromosome 21 open reading frame 29 3.6 XM_002169621 SCO1-like, putative 3.6 XM_002163648 hypothetical protein LOC100198744 3.5 XM_002171093 Zinc finger BED domain-containing protein 1 3.5 XM_002159881 Pftaire-1 3.1 XM_002157841 DAZ-like protein 3.1 XM_002170387 kelch-like 2, Mayven 3 XM_002159143 ATP-binding cassette, sub-family B (MDR/TAP), member 6 2.9 XM_002159705 tetratricopeptide repeat domain 18 2.9 XM_002160702 predicted protein (LOC100211463) 2.9 XM_002158823 antistasin 2.9 XM_002167903 transposase (putative) 2.8 XM_002157873 hypothetical protein LOC100213162 2.7 XM_002169812 Os02g0236500 (LOC100204594) 2.7 XM_002166284 nuclear transcription factor Y, gamma 2.6 XM_002154859 conserved hypothetical protein (LOC100211478) 2.5 XM_002162162 predicted protein (LOC100210397) 2.5 XM_002159819 hypothetical protein LOC100200934 2.5 XM_002154283 predicted protein (LOC100211827) 2.4 XM_002165420 hypothetical protein LOC100205972 2.4 XM_002155669 hlpA 2.4 XM_002157447 ADP-ribosylation factor-like 11 2.4 XM_002166137 DNA damage-binding protein 1 2.4 XM_002155169 PIF/Harbinger-like protein 2.4 XM_002165417 Aldehyde dehydrogenase type III 2.4 XM_002162621 predicted protein (LOC100206788) 2.4 XM_002167112 predicted protein (LOC100212131) 2.3 XM_002165502 viral A-type inclusion protein 2.3 XM_002170817 hypothetical protein LOC100200832 2.2 XM_002159385 gamete fusion-like protein (FusM) 2.2 XM_002168770 predicted protein (LOC100212392) 2.2 XM_002154967 Plastid ribosomal protein L11 large ribosomal subunit 2.2 XM_002165246 Dihydrolipoyl dehydrogenase, mitochondrial 2.1 XM_002154102 conserved hypothetical protein (LOC100207416) 2.1 XM_002171100 LINE1-like RNA-directed DNA polymerase 2.1 XM_002160768 small heat shock protein 2 XM_002162396 predicted protein (LOC100212840) 2 XM_002169690 RCC2 homolog 2

Acetaminophen (5,000 μg/L) gene with reduced expression in the 72-hour exposure group (3 types) GenBank Acc. Gene name Expression change XM_002163542 thrombospondin type 1 repeat-containing protein 2 -3 XM_002169302 hypothetical protein LOC100206448 -4.7 XM_002169831 hypothetical protein LOC100209805 -5

25 genes selected from differential expression of acetaminophen GenBank acc. Gene symbol 12 hours 24 hours 48 hours 72 hours Sequence number 1.DNA damage XM_002170132 PMS1 2.3 One XM_002166137 DDB1 2.4 2 XM_002163049 MUC1 2.8 3 XM_002166161 MUC2 4.4 4 2. Protein damage XM_002165146 TPR/ uncharacterizedLOC100207207 3.1 5.2 5 XM_002170387 KLHL2 3 6 3. Genomic instability XM_002154329 PIF1 4.2 3.3 4.4 7 XM_002155669 HLPA 2.4 8 XM_002165417 ADLH3 4.1 2.4 9 XM_002162252 APX 2.2 15.1 10 XM_002158049 GSTM2 3.1 33.6 11 XM_002165246 DLD 2.1 12 XM_002155491 Oxidative stress protein -2.5 -2.8 -2.6 13 5. Stress response XM_002170619 KPI 3.5 18.1 14 6. Apoptosis XM_002157447 ARL11 2.4 15 7. Cell cycle XM_002169690 RCC2 2.6 2 16 XM_002159881 PFTAIRE1 3.1 17 8. Sex determination XM_002166349 CNVAS1/RH37 2.8 2.1 18 XM_002159385 FUSM/HAP2A 2.4 2.1 2.6 2.2 19 XM_002161814 CNNOS1/ 3.7 20 uncharacterized LOC100192236 XM_002157841 DAZL 2.2 3.1 21 9. Morphogenesis XM_002155086 FSCN2/Fascin-like 2.8 10.3 22 XM_002159360 NR2F2 2.8 23 10. Multidrug resistance XM_002159143 ABCB6 3.6 2.9 24 11.Immune sysyem XM_002159617 CDK5RAP1 9.6 25

In addition, as shown in Table 10, among the differentially expressed genes, DNA damage, protein damage, genomic instability, oxidative stress response, and stress response ), 25 species expected to be involved in apoptosis, cell cycle, sex determination, morphogenesis, multidrug resistance, and immune system Genes were selected (Table 10).

Accordingly, these 25 selected genes can be usefully used as biomarkers that can predict acetaminophen exposure in aquatic ecosystem environments and predict physiological and metabolic changes of hydra by exposure to acetaminophen.

<110> Korea Institute Of Ocean Science & Technology <120> Acetaminophen responsive genes in hydra magnipapillata and the method for diagnosing aquatic environment pollution using the same <130> 2020p-01-015 <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 1278 <212> RNA <213> Hydra magnipapillata <400> 1 atgaataagt tacctttatc tacagtagaa cttattacaa gcactcaggt tataatcagt 60 ttggtgtctg ttgtaaaaga acttgttgaa aactcacttg attctggctg tgacagcata 120 gatgttagat tggatgaaaa tggtctatct aagattgaag tgcaagataa tggtactggt 180 attgctttag aagatattca atatgtttgc aaacagcatt acacaagcaa aattagaaat 240 gaattagatt tgcttaactt aacgcaatat ggttttcgtg gtgaagctct tttttcaata 300 tgttcagttg gtgatgtttt tataacaact aaaacaaaag cagatcaggt ttcaaaaaaa 360 tatagttttt caaagtatgg tgaaattatt agttctgaaa taactcatca taatgtagga 420 actatagtca ctgtattaaa tttgttcaaa gcattgcctg ttcgcagaaa attatatgag 480 aacaatcgta aagttagaga agaagttaag tgtattgaag agttactaat atcttacggc 540 atagcgatgc ccaaactgcg cattactctt aagtatgaca aaaatataat atggaggaaa 600 gatcgtgctg ctaattgttc attagttttg tctgcattat ttggttctaa tgttttaaat 660 aatctaaagg agatagaata taaagatgaa gaattacaag taaaagctta tatacctaaa 720 gttggttctg cttcaaatgt ggttggtaga actcttgcag atagaatgtt tataattgta 780 aataaaagac ctgttagatc aaaagaaatc aagtctctat taaaaacttc ttacaacaat 840 aattgttgcc aaggaaaata tcctttctca gttcttatga tagaagtaaa accagaccaa 900 attgatgtaa atcttcatcc aaacaaaatg gatatccttt tacaaaatac tgtgagtata 960 ttaaatagtg caatgagcat atttagcgac atttctgcaa gagatatttt taaacttgtt 1020 cttggtgagc ttaaggaagt ttgggaaaaa gctggagtac caattcgatc agataactct 1080 tgtcattcat tgcttaccaa attgtttgtg ggaaaagtga aaataaaaaa aattgatcac 1140 ccaagtcgag atgttaaagc tggaaaagaa aaaattgtca gattttgtaa tgaacttgag 1200 caactttgcg acatttcagc agtaaatgca tatgaacagt tattagtatc acgtagacca 1260 aaatggaaag aggattag 1278 <210> 2 <211> 377 <212> RNA <213> Hydra magnipapillata <400> 2 tttgtgataa taagcatcac atattgaata attaaagttt ttcagaaaaa gaaaacatgg 60 catataacta tgttgttaca gcacaagagg caactgcgat caatgctaca gtaactggtc 120 actttacagg accaacagac ttaaatttga tagttgctaa aaataataaa ttagagttac 180 atatggtaac tcctgagggt ctgcaaccta aatttgattt aaatgtgtat gggagagtag 240 cagttatgca attatttaga cctcaaaatg aaaaccaaga tttgcttttt atattaactg 300 agcggtatcg aactgctata ttagcttata aagcagaaac tggtgatatt ataacaaaag 360 cttatggtga tgtgcag 377 <210> 3 <211> 1041 <212> RNA <213> Hydra magnipapillata <400> 3 atggttaaga aatatagtgg catgaatgca ggattgagag aaacagatcg taactgtatt 60 ttttctagct gtgtaaatcg tgctgtaaat cttgtgaatg tcgattgcgc tcaatcgaca 120 atgaagcaat tgcgtagttt ggaaaaactc aacaaatcac taccactcgc accaaatttt 180 tcaacagcac ttccaatcgc accaaatctt tctacaacaa taccaatcgc accaaatctt 240 ttaacagcac ttccaaccgc accaaatctt tcaacagcac ttccaatcgc accaaatctt 300 tcaacagcac ttcctatcgc accaaatctt tcaacagcac ttccaatcgc accaaatctt 360 tcaacagtac ttccaatcgc accaaatctt tcaacagcac ttccaatcgc accaaatctt 420 tcaacagcac tactaatcgc accaaatctt tcaacagcac taccaatcgc accaaatctt 480 tcaacagcac taccaatcgc accaaatctt tcaacagcac taccaatcgc accaaatctt 540 tcaacaacac taccaatcgc accaaatctt tcaacagcac tttcaatcgc accaaatctt 600 tcaacagcac taccaatcgc accaaatctt tcaacagcac taccaatcgc accaaatctt 660 tcaacaacac taccaatcgc accaaatctt tcaacagcac tttcaatcgc accaaatctt 720 tcaacagcac taccaatcgc accaaatctt tcaacagcac ttccaatcgc accaaatctt 780 tcaacagcac tttcaatcgc accaaatctt tcaacagcac taccaattgc accaaatctt 840 tcaacagcac ttccaatcgc accaaatctt tcaacagcac ttccaatcgc accaattctt 900 tcaacagcac taccaatcgc accaaatctt tcaacagcac ttccaatcgc accaaatctt 960 tcaacagcac taccaatcgc accaaatctt tcaacagcac taccaatcgc accaaatctt 1020 ggcaataagt tttcatttta a 1041 <210> 4 <211> 10227 <212> RNA <213> Hydra magnipapillata <400> 4 atgtttacag gactcatatc tcttcaaaca actcaattaa ttattccaat tagagaagta 60 aatttaattt caaatatttc attgtcaaat gaaattaata aaggacaact atcgcttgaa 120 acaactttaa atgaaataaa cacttccatt tccattaaaa aaacaaatca ggaaacaaat 180 caaccaataa ccggaacatt aaaaacaaca acatctaaaa cacaaataaa cacaaacgaa 240 gcatcaccaa tagaagaaac aacaactatt gtaaacactg tagcagctac agacacactt 300 aaaactactc catctgaagt tcaaacaagc acaaacacag tatcaccaat agaagaaaca 360 accacattac ccagtactga cgcagttaca gacacagttg aaactactcc aaataaagtt 420 caaacaagca caaacatagc atcaccaata aaagaaataa caacattacc cagtactgat 480 gcagtaaaag acacaattga aagtactcca tctaaagtta aaacagacac aaacacagca 540 tcaccaataa aagaaacaac aattattgta agtactgcag cagctacaga aacagttgaa 600 actactccat ctgaagttca aacaactaca aataaaacat caccattaga agaaacaaca 660 actattgtaa gtactgcagc agctacagac acagttgaaa ctactccatc tatagttcaa 720 acaagcacaa acacagcatt accaatagaa gaaacaacaa cattactcag tactgatgca 780 gttacagaca cagttaaaac tactccattt gaagttcaaa caaccacaaa taaagcatca 840 ccaataaaag aaacaaccac attacccagg actaatgcag ttaaagacac agttaaaact 900 actccatctg aagttcaaaa aatcacaaac acagcatcac caattgaaaa aaaaacaact 960 attgtaagca ctacagcagc tataggcact gttaaaacta ctccttctga agttcaaaca 1020 agcacaaaca cagcatcacc aatagaagaa acaacaacat tacccagtac tgatgcagtt 1080 acagacacag ttgaaactac accatctgaa gttcaaacaa gcacaaacac agcatcacca 1140 atagaagaaa caacaactat tgtaagtact acagcagcta cagaaacaat tgaaactact 1200 ccatctgaag ttcaaacaag cacaaacaca gcatcacaaa tagaagaatc aacaactact 1260 gtaagcactg cagcagctac agacacagct gaaactactc catctgaagt tcaaacaacc 1320 acaaatacag catcaccaat agaagaaaca acaactatcg taagtactgc agcagctaca 1380 gaaacagttg aaactactcc atctgatatt caaacaacca caaatacagc atcaccaata 1440 gaagaaacaa caactattgt aagtacttca gcagctacag aaacagttga aactactcca 1500 tctgaagttc aaacaagcac aaacacagca tcacaaatag aagaaacaac aactattgta 1560 agcactgcag cagctacaga cacatttgaa actactccat ctaaagttca aaaaagcaca 1620 aacacagcat cagcaataga agaaacaaca actattgtaa ttactgcagc agctacagaa 1680 acagttgaaa ctactccatc taaagttcaa acaaccacaa atacagcatc accaatagaa 1740 gaaacaacaa ctatcgtaag tactgcagca gcaacagaca cagttgaaac tactccatct 1800 gaagttgaaa taaccacaaa tacagcatca ccaatagaag aaaaaacaac tattgtaagt 1860 acttcagcag caacagaaac agttgaaact actccatctg aagttcaaac aagcacaaac 1920 acagcatcac aaatagaaga aacaacaact attgtaagca ctgcagcagc tacagacaca 1980 tttgaaacta ctccatctga agttcaaaca accacaaata cagcatcacc aatagaagaa 2040 acaacaacta ttgtaagaac ttcagcagct acagaaccag ttgaaactaa tccatctaaa 2100 gttcaaaaaa gcacaaacac agcatcagca atagaggaaa caacaactat tgtaattact 2160 gcagcagcta cagaaacagt tgaaactact ccatctgaag ttcaaataac cacaaataca 2220 gcatcatcaa tagaagaaac aacaactatt gtaagtactt cagcagctac agaaacaatt 2280 gaaactactc catctgaagt tcaaacaagc gcaaacgcag catcagcaat agaagaaaca 2340 acaactattg taagtactgc agcagctaca gaaacagttg aaactactcc atctgaagtt 2400 caaacaagca caaacacagc atcacaaata gaagaaacaa caactattgt aagcactaca 2460 gcagctatag acactgttaa aactactcca tctgaagttc aaacaagcac aaacacagca 2520 tcaccaatag aagaaacaac aacaattgta agtacatcag aagctacaga aacagttgaa 2580 actactccat ctgaagttca aacaagcaca aacacagcat cacaaataga agaaacaaca 2640 actattgtaa gcactgcagc agctatagac acagttgaaa ctactccatc tgaagttcaa 2700 acaaccacaa atacagcatc accaatagaa gaaacaacaa ctattgtgag tacttcagca 2760 gctacagaca cagttgaaac tactccatct gatgttcaaa caaccacaaa tacagcatca 2820 ccaatagaag aaacaacaac tattgtaagt acttcagcag cttcagaaac agttgaaact 2880 actccatctg aggttcaaac aagcacaaac acagcatcag caatagaaga aacaacaact 2940 attgtaagta ctgcagcagc tacagaaaca gttgaaacta ctccatctaa agttcaaaca 3000 agcacaaaca cagcatcacc aatagaagaa acaacaacta ttgtaagtac tgcagcagct 3060 acagaaacag ttgaaactac tccatctgaa gttcaaacaa gcaccaacac agcatcacca 3120 attaaaaaaa taacaactat tgtaagcact acagcagcta gaggcactgt tgaaactact 3180 cctactgaag ttcaaacaag cacaaacaca gcatcacaaa tagaagaaac aacaactatt 3240 gtaagcacta cagcagctat agacactgtt aaaactactc catctgaagt tcaaacaagc 3300 acaaacacag catcaccaat agaagaaaca acaactattg taagtacttc agaagctaca 3360 gaaacagttg aaactactcc atctgaagtt caaacaagca caaacacagc atcaccaata 3420 gaagaaacaa caactattgt aagcactgca gcagctacag acacagttga aactactcca 3480 tctgaagttc aaacaagcac aaacacagca tcaccaatag aagaaacaac aactattgtg 3540 agtacttcag cagctacaga cacagttgaa actactccat ctgaagttca aacaaccaca 3600 aatacagcat caccaataga agaaacaaca actattgtaa gtacttcagc agcttcagaa 3660 acagttgaaa ctactccatc tgaggttcaa acaagcacaa acacagcatc agcaatagaa 3720 gaaacaacaa ctattgtaag tactgcagca gctacagaaa cagttgaaac tactccatct 3780 gaagttcaaa caaccacaaa tacagcatca ccaatagaag aaacaacaac tattgtaaga 3840 acttcagcag ctacagaaaa agttgaaact actccatctg aagttcaaac aagcacaaac 3900 acagcatcag caatagaaga aacaacaact attgtaagta ctgcagcagc tacagaaaca 3960 gttgaaacta ctccatctga agttcaaaca agcacaaaca cagcatcaca aatagaagaa 4020 acaacaacta ttgtaagcac tacagcagct atagacactg ttaaaactac tccatctgaa 4080 gttcaaacaa gcacaaacac agcatcacca atagaagaaa caacaactat tgtaaatact 4140 tcagcagcta cagaaacagt tgaaactact tcatctgaag ttcaaacaag cacaaacaca 4200 gcatcacaaa tacaagaaac aacaactatt gtaagcactg cagcagctac agacacagtt 4260 gaaactactc catctgaagt tcacacaacc acaaatacag catcaccaat agaagaaaca 4320 acaactattg taagtacttc agcagcttca gaaacagttg aaactactcc atatgaggtt 4380 caaacaagca caaacacagc atcagcaata gaagaaacaa caactattgt aagtactgca 4440 gcagctacag aaacagttga aactactcca tctgaagttc aaacaagcac aaacacagca 4500 tcacaaatag aagaaacaac aactattgta agcactgcag cagctacaaa cacagttgaa 4560 actactccat ctgaagttca aacaaccaca aatacagcat caccaataga agaaacaaca 4620 actattgtaa gtacttcagc agctacagaa acagttgaaa ctactccatc tgaagttcaa 4680 acaagcacaa acacagcatc agcaatagaa gaaacaacaa ctattgtaag tactgcagca 4740 gctacagaaa cagttgaaac tactccatct gaagttcaaa caagcacaaa cacagcatca 4800 caaatagaag aaacaacaac tattgtaagc actacagcag ctatagacac tgttaaaact 4860 actccatctg aagttcaaac aagcacaaac acagcatcac caatagaaga aacaacaact 4920 attgtaagta cttcagcagc tacagaaaca gttgaaacta ctccatctga agttcaaaca 4980 agcataaaca cagcatcaca aatagaagaa acaacaacta ttgtaagcac tgcagcagct 5040 acagacacag ttgaaactac tccatctgaa gttcacacaa ccacaaatac agcatcacca 5100 atagaagaaa caacaactat tgtaagtact gcagcagcta cagaaacagt tgaaactact 5160 ccatctgaag ttcaaacaac cacaaataca gtatcaccaa tagaagaaac aacaactatt 5220 gcaagtactt cagcagctac agaaacagtt caaactactc catctgaagt tcaaacaagc 5280 acaaacacag catcaccaat tgaaaaaata acaactattg taagcactac agcagctata 5340 ggcactgttg aaactactcc atctgaagtt caaacaagca caaacacagc atcaccaata 5400 gaagaaacaa caacattacc caatactgat gcagttacag acacagttaa aactactcca 5460 tttgaagttc aaacaaccac aaatacagca tcaccaatag aagaaacaac aacattaccc 5520 agtactgatg cagttacaga cacagttgaa actacaccat ctgaagttca aacaagcaca 5580 aacacagcat caccaataga agaaacaaca actattgtaa gtactacagc agctacagaa 5640 acaattgaaa ctactccatc tgaagttcaa acaagcacaa acacagcatc agcaatagaa 5700 gaaacaacaa ctattgtaag tactgcagca gctacagaaa cagttgaaac tactccatct 5760 gaagttcaaa caagcacaaa cacagcatca caaatagaag aaacaacaac tattgtaagc 5820 actacagcag ctatagacac tgttaaaact actccatctg aagttcaaac aagcacaaac 5880 acagcatcac caatagaaga aacaacaact attgtaagca ctacagcagc tatagacact 5940 gttaaaacta ctccatctga agttcaaaca agcacaaaca cagcatcaca aatagaagaa 6000 acaacaacta ttgtaagcac tgcagcagct acagacacag ttgaaactac tccatctgaa 6060 gttcaaacaa gcacaaacac agcatcacca attgaagaaa caacaattat tgtaagtact 6120 tcagcagcta cagaaacagt tgaaactact ccatctgaag ttcaaacaag cacaaacgca 6180 gcatcacaaa tagaagaaac aacaactatt gtaagcactg cagcagctac agacactgtt 6240 gaaactactc catctgaagt tcaaacaagc acaaacacag catcaccaat agaagaaaca 6300 acaactattg taagtactga tgcagcttca gacacagttg aaactactcc atctgaggtt 6360 caaacaagca caaacacagc atcaccaata gaagaaacaa caactattgt aagtactgca 6420 gcagctacag aaacagttga aactactcca tctgaagttc aaacaagcac aaacacagca 6480 tcacaaatag aagaaacaac aactattgta agcactgcag cagctacaga cacagttgaa 6540 actactccat ctgaagttca aacaaccaca aatacagcat caccaataga agaaacaaca 6600 actattgtaa gtacttcagc agctacagaa acagttaaaa ctactccatc tgaagttcaa 6660 acaagcacaa acacagcatc agcaatagaa gaaacaacaa ctattgtaag tactgcagct 6720 gctatagaaa cagttgaaac tactccatct gaagttcaaa caagcacaaa cacagcatca 6780 caaatagaag aaacaacaac tattgtaagc actacagcag ctatagacac tgttaaaact 6840 actccatctg aagttcaaac aagcacaaac acagcatcac caatagaaga aacaacaact 6900 attgtaagta cttcagcagc tacagaaaca gttgaaacta ctccaactga agttcaaaca 6960 agcacaaaca cagcatcaca aatagaaaaa acaacaacta ttgtaagcac tgcagcagct 7020 acagacacag ttgaaactac tccatctgaa gttcaaacaa gcacaaacac agcatcacaa 7080 atagaagaaa caacaactat tgtaagcact gcagcagcta cagaaacagt tgaaactact 7140 ccatctgaag ttcaaacaag cacaaacaca gcatcacaaa tagaagaaac aacaactatt 7200 gtaagcactg cagcagctac agacacagtt gaaactactc catctgaagt tcaaagaacc 7260 acaaacacag catcaccaat agaagaaaca acaactattg tgagtacttc agcagctaca 7320 gacacagttg aaactactcc atctgaagtt caaacaacaa caaatacagc atcaccaata 7380 gaagaaacaa caactattgt aagtacttca gcagcttcag aaacagttga aactactcca 7440 tctgaggttc aaacaagcac aaacacagca tcagcaatag aagaaacaac aactattgta 7500 agtactgcag cagctacaga aacagttgaa actactccat ctgaagttca aacaagcaca 7560 aacacagcat cacaaataga agaaacaaca actattgtaa gcactgcagc agctacagac 7620 acagttgaaa ctactccatc tgaagttcaa acaaccacaa atacagcatc accaatagaa 7680 gaaacaacaa ctattgtaag tacttcagca gctacagaaa cagttaaaac tactccatct 7740 gaagttcaaa caagcacaaa cacagcatca gcaatagaag aaacaacaac tattgtaagt 7800 acagcagctg ctacagaaac agttgaaact actccatctg aagttcaaac aagcacaaac 7860 acagcatcac aaatagaaga aacaacaact attgtaagca ctacagcagc tatagacact 7920 gttaaaacta ctccatctga agttcaaaca agcacaaaca cagcatcacc aatagaagaa 7980 acaacaacta ttgtaagtac ttcagcagct acagaaacag ttgaaactac tccaactgaa 8040 gttcaaacaa gcacaaacac agcatcacaa atagaaaaaa caacaactat tgtaagcact 8100 gcagcagcta cagacacagt tgaaactact ccatctgaag ttcaaacaag cacaaacaca 8160 gcatcaccaa ttgaaaaaat aacaactatt gtaagcacta cagcagctat aggcactgtt 8220 gaaactactc catctgaagt tcaaacaagc acaaacacag catcaccaat agaagaaaca 8280 acaactattg taagtattac agcagctaca gaaacaattg aaactactcc atctgaagtt 8340 caaacaagca caaacacagc atcagcaata gaagaaacaa caactattgt aagtactgca 8400 gcagctacag aaacagttga aactactcca tctgaagttc aaacaagcac aaacacagca 8460 tcacaaatag aagaaacaac aactattgta agcactacag cagctataga cactgttaaa 8520 actactccat ctgaagttca aacaagcaca aacacagcat caccaataga agaaacaaca 8580 actattgtaa gcactacagc agctatagac actgttaaaa ctactccatc tgaagttcaa 8640 acaagcacaa acacagcatc accaatagaa gaaacaacaa ctattgtaag cactgcagca 8700 gctacagaca cagttgaaac tactccatct gaagttcaaa caagcacaaa cacagcatca 8760 caaatagaag aaacaacaac tattgtaagc actgcagcag ctacagacac agttgaaact 8820 actccatctg aagttcaaac aagcacaaac acagcatcac caattgaaga aacaacaatt 8880 attgtaagta cttcagcagc tacagaaaca gttgaaacta ctccatctga agttcaaaca 8940 agcacaaacg caatatcaca aatagaagaa acaacaacta ttgtaagcac tgcagcagct 9000 acagacactg ttgaaactac tctatctgaa gttcaaacaa gcacaaacac agcatcacca 9060 atagaaaaaa caacaacatt acccagtact gatgcagtta cagacacagt tgaaactact 9120 ctatttgaag ttcaaacaac cacaaataga gcatcaccaa taaaagaaac aacaactatc 9180 gtaagtacat cagcagctac agaaacagtt gaaactactc catctgaagt tcaaacaagc 9240 acaaacacag catcaccaat tgaaaaaata acaactattg taagcactac agcagctata 9300 gacactgttg aaactactcc atctgaagtt gaaataagca caaacacagc atcaccaata 9360 gaagaaacaa caactattgt tagtactaca gcagctacag aaacagttga aactactcta 9420 tctgaagttc aaacaagcac aaacacagca tcacaaatag aagaaacaac aactattgta 9480 agcactgcag cagctacaga aacagttgaa actactccat ctgaaattca aacaagcaca 9540 aacacagcat taccaataga agaaacaaca actattgtaa gtacttcagc agctacagaa 9600 acagttgaaa ctactccatc tgaagttcaa acaagcacaa acacagcatc acaaatagaa 9660 gaaacaacaa ctattgtaag cacttcagca gctacggaca cagttgaaac tactccatct 9720 gaagttcaaa caagcacaaa cacagcatca ccaagacaag aaacaacagt taaactacaa 9780 acaacaactc taaaattttc atctaaaatt gctcctacac caatagcgcc tccatgccct 9840 cagaaacttt gtcaaggctt gactgatgga aattatgaaa ttctaggata tccccatgat 9900 tttgttcagt gttctaatgg aattgcttct tgtcaatctt gctggcctca aagtttagtt 9960 tttagtcaat cttgcaatca atgtttatac aataaagaag attcctgcta tgcagagaag 10020 gcgtggacgc cgcaatctac ttatccgtgt ccagatgcct gtagtagtaa agggccaaat 10080 ttttcgggta acattgctga tagaacttct aacaaaggat cagggaatcc ttttcattac 10140 attgggtgtt ggaatgggat tactcaagga tgtgttacat gcccatcagg tttaaaattc 10200 aacgaagagc agaacgcatg tttataa 10227 <210> 5 <211> 717 <212> RNA <213> Hydra magnipapillata <400> 5 atcgtattat caattcaatt ctcatttaaa ttgtatttat aactcaaggg aataatttta 60 attagttaac ttattaggca actttgtgtt attaaaacta taaagattta tgtttgtagc 120 attttattta ccaaatttat ataaagattt cgcaagattt acttaaatta tttattttga 180 aaaatcgttt aaatttgcaa gtttagtttt tttatcaagc accaaactcg atgactaaca 240 aacgcaagtt tgtgagagaa gaaaaggttg aagcagacag tgataagatt ggaaatgtag 300 aagacgaggt accgaagcaa ctagaagaaa ctgatcataa cttatccaca gatttagata 360 aaccgctgag ctctttaaca tctgaatcga taattaatca tgtgcaatct ataattgatg 420 atatggaagt caaaagaaaa gatgacgaaa ccctaatcgt agagtttcga aaatctatgg 480 agatacaaac tgaaatctgg tgtgatcttt tggaaaagac acttgcaaaa gtttacatga 540 aaaataacaa tacttgtcaa gaaaagtttc aacagctgca taccatatta ggacgaataa 600 gtcagcttga acaagaaatg tcttctttta aacaatcttt aaattcattg tatgctgaag 660 tacaagctac ctatccatca ttgcagtagc aagcataaac tattttaatt tttaatt 717 <210> 6 <211> 460 <212> RNA <213> Hydra magnipapillata <400> 6 agcaactcta aagtaaacaa acaaaaacta cacataatgt gtgtgttgct tcagatatat 60 agaaaacatg gcgtttttat cacataaata tacaggcagt tgttctggtt ttagtttacc 120 tatatttaag aacccattgc atttaacaaa atcatttgac attctctatg caatgtggaa 180 acaaaattta ctttgtgatg tcaccatatg tgttggaaac aaatctatcg aagctcatag 240 ggcaatactt gcctcttcca gtccttactt ttatgctatg ttcacaggag agatgtcaga 300 aagtaaacag aactgcatag ttttaaaaga agttgatgca tatgctttag agctgcttat 360 aggttattgc tattcaggag aggtgcaagt tactgaagaa aatgttcaag tgaagaagtc 420 cgccattatg gattttgtag ttgtactttt aattgaataa 460 <210> 7 <211> 1899 <212> RNA <213> Hydra magnipapillata <400> 7 atgactttat cagctaaaca aaaattggct tttcaatggt ttgacgaagg attaaatttt 60 tttaatactg gtagtgctgg ttgtggtaaa agttatatac ttgatgctat tgcttctagc 120 gatcaaatat ttaaaaatat tgcagtaaca gctagtacag gaaaagcagc tcatcagatt 180 aagggtatga ctgtgcaggt tttagaaaga tggagaaata cacatgtttt gattattgat 240 gaaatttcaa tgcttaacgc agaaaccttt gatttattac atcatttagc atgtaaaata 300 aatcaatgca atgacgaatt gtttggtggg atacaagtta ttacatgcgg tgatttccgt 360 caaatgccac ctgtaaaagg tgaatatgtt ttcaagtctg caatatggaa aaaatacatg 420 tttaatgtta ttgaacttac tgaatctttt agacaagaaa atattgaatt ttttaacgct 480 ttaaacgaaa tacgtattgg aaaagtatct gataaaacgt taatgagtat tccttctgat 540 gaacgttggt ttcattctaa agatgttatt aaaaatttaa aaataaatcc attgtttcaa 600 atacctatta gtataaatat taaaattggc gctgtagtca tgttagttaa aaattttaac 660 gttgaaggtt tatgcaacgg tacaattggt attgtaacat ttatagaaac agatggtgtt 720 tgggttaata taaacggtag agaatttaaa atcgaaaacg ttagagaaga cattttagat 780 tgtactcata gtattattgc atctagaatc ggtttacctt tacaattagc attttcttta 840 actgttcata aagcgcaagg ttgcacacaa atattgtggc aaaaattgga tgcaaatgac 900 gaaaatgttg aaaaatataa aagcgaactg agagtatata atttgttagt tttggctgac 960 actttagttc gttttaccta cgaaaaatgt ttacctcgtt ttcaaaattt aaacctttct 1020 gaacaggaaa aatgtcaagt atggcaagaa gaaaatcctt tgtgtacaat atgcagatgt 1080 cctgcagaca aaataaagaa atctgatgtg tacagttaca aattaactac aatatggtgg 1140 gaaaaatttg accgcactct tataacaaac gggtttttaa gtgattatgt aagaataaac 1200 tcgttagatc tagtcgatca catgaaatgt ttaagtcaac tcgttatgaa cgagcaacaa 1260 attaacatat ggtttgaaaa tgatcctata gaattactag aagattctta ttttttaata 1320 cgaatttttc aaaaatgtat caacgaagtt aacgtgcatt ggaaatatga acagtcttcg 1380 agccagcttg attcttccat tcaacactac tcttcttgcg aactttattc ttccagtcag 1440 cttgattctt tcagtgaatt tgattcttct tcaagtagcg aagaaaatgt agtagaaaca 1500 gatgtgacaa ataatgagct actttctctt ttaagatgca gtggacattc agctttagct 1560 aaatcttctc gtactttgtt aaagactcaa cgctgtgttt tgatagaaaa aaagtgtggt 1620 ggggagtata tttatttagg gttaaggaat ggtattagtc gaactttcag agagaatcct 1680 ttattatcaa ttgagaacaa catcatatct ttattgatgt caatgaaaga tttatgggca 1740 ttagttgtaa cttcaagcaa agacaaaatt ggaaaaatta gagagacgga aacagttgtt 1800 ccatttcatt ggatttcctc agatgagaaa tggttgtttt ggcctccttt tcgaaaagat 1860 gaaaaatata caaaggcaat tcccaatgaa gttacttga 1899 <210> 8 <211> 285 <212> RNA <213> Hydra magnipapillata <400> 8 atgaacaaag gcgaattgat cgaagcattg gctgcaaagt ctgaactgtc caaagctgct 60 gccggacgaa ctctggatgc tttgattgag atcatcactg ccaaagtggc aaaaaaagaa 120 gatgtgcaat tgattggctt tggcactttc aaggctgcca agcgtgctgc acgcactggg 180 aagaacccac gtactggtga agcactgaag attactgcga cgacagtgcc caagttcaca 240 ccgggtgcag ccttcaaggc agctgttgcc aagaagaaaa agtaa 285 <210> 9 <211> 936 <212> RNA <213> Hydra magnipapillata <400> 9 atgggcaaca gcgcccatgt gcagtaccag ccccgcggcc gctgcctgat catcgcgccc 60 tggaactacc ccctgtacct gatgctcagc ccgctggtct ccgccctagc cgccggcaac 120 acggccatcc tcaagccctc tgagatggcg ccgcgcgtgg cccaagtgct ggctagcatc 180 gtggccaagg ccttcccggc gcatgaagtg gctctgtttg aaggggcact ggccacctcg 240 caggccttgc tggccatgcc gtttgaccac atcttcttca ccggctcacc cgccgtgggc 300 aaggtggtca tggcggctgc ggccaagaac ctgaccagcg tgaccctcga gctgggcggc 360 aagtccccca ccatcgtgga cgagaccgcc gacctgcaaa aggcggctga aaccattctg 420 tggggcaagt ttgtcaacgc cggccagacc tgtgtggcgc ccgactacct gtatgtggac 480 accgccggta actacattgc cccgaccctg gtcgacaacg tgcccatgga ctccaccctg 540 atgcaggagg aaatcttcgg cccggtgctg cccatcattc cctacaccga cctgcagcag 600 gtcatcacct ccatcaatgc cgaccagaag ccactggccc tgtatatctg gagcagcaac 660 cagagcaaca tcgacaccgt gctcaccaac accagctccg gcggcgcctg cgtgaatcac 720 tgcgtgctgc acgtggccca cggcaacctg cccttcggcg gggtcaacaa ctccggcatc 780 ggcagctcgc acggcattta cgggttcaag gccttctcgc atgaacgtgc ggtgctcaag 840 ggcggctggc tgcccagcat ccgcatgttc ttcccgccct acacagcggg ccgcaccaag 900 ctgctgaact tcttggtgag ctgggtcagc cgatag 936 <210> 10 <211> 1183 <212> RNA <213> Hydra magnipapillata <400> 10 caaaaaatat tatttttgtt ataaaattta acactaaact gatcagacgc ccttgttagc 60 tattagtttc aacgcaacaa tgaagaacaa aatagtttgg tcaacaatag cgtatttgct 120 aacttttttt ttgttcgcta attcagttca aattattcct tcaagttatt attttgaaaa 180 agcaaaaagc gatctattaa atctaattga atctgttaaa aatggtaatg acttaccaat 240 gattgctggc actgttcgtg tagcgtttca cgattgcatt ggtaaaggaa aatgtaacgg 300 ttgtatagat cattcattat catccaacag agggttaaaa agagtaacgg atcgacttga 360 tgcattatac gatgtttctt acaaaggaaa aatgtcaaga gcggattttt atgcattagc 420 agcagttatt gctctaacta ggtctacagc caatgttcct gataaataca atgggctcaa 480 aacttttaaa gtgggcagaa aggattgtga tacgtcacct gttgattctg aaaccaattc 540 caattttcca agaggaagcg atggcacgtc aaaaacatta gggtttttta aaagggagtt 600 tggattcaac actagagagg ctgttgctat tttaggtgcg cacacacttg gaagatgtca 660 tttgcaaaac tcaggatttg ttggtgcctg ggttgataat cgattttcaa cagcgttgcc 720 gggtgaaact ttggctccaa caagtatact cgataatgct tactttcgaa tgattgttga 780 tatagttccg tgggttcagg tgaacatcaa tggaacaaga atccaatggc aagaaccaaa 840 acatccaata cctaatgaca agttgccaga atcgaaaaga agtccattac ttttaaactc 900 tgatatgagt atttcatgga ttataacacc cacagataat cttggtacag tttcttgtac 960 accaacctca cgaataaatc cttgccgcca ttctaatgct catacatatg caaaactcta 1020 cgcccaaaat aatgcatttt ggataaaaga gtttacaaga gtttttaata aaatgataag 1080 tagaagtgaa tacacgttaa aagaggttcc cgaaacaaac ttatacgaag atttgtactt 1140 taataataga aaagtgtaca atgatgccga agaagtattt taa 1183 <210> 11 <211> 597 <212> RNA <213> Hydra magnipapillata <400> 11 acttgtcaat tatgtttttt tacaaatgct ttaaaatgag cattttttta aaattgaaac 60 ataaacgttg aagttgattt tggttgttta ataaaaatta tatttatatc aatacattgc 120 gtttatatat ttacagttta aaagatgaca actcctacac ttggttactg gagaattaga 180 gggctcgctc agccaattcg tttactactt ggctatacaa aaacagaatt tgaagacaag 240 atgtatgatg ttggagatgc accaacttat gatcgatcag catggacaga cgtaaaatat 300 acactcgggt tagatttacc aaacttgcct tattttattg atggcgatat taaattaacc 360 atgagcaatg ctattcttcg ttatattgca agaaagaaca acatggttgg aaacacagag 420 cacgagattt gtagagtgga tatgatggag aatgaggcat ttgtgaaaag atttgaggaa 480 ttaccagaaa tacacgaatt catgagttca tacaaattct ttaaaggtcc cccaaacgga 540 aaaatggctg tcattggtgg agagttacaa aatacacaat attgtttacc gcaataa 597 <210> 12 <211> 1809 <212> RNA <213> Hydra magnipapillata <400> 12 atggcattgg tagaagtaaa agtcccggat atcggtgact tcgacgaagt cgcggtcatc 60 gaactgctgg tcaaagtggg cgacaccgtg aaggtggagc aaagtctgat taccgtggag 120 agcgacaagg cttccatgga gattccttcc agcaccgccg gtgtggtgaa ggaaatcaag 180 gtcgccctgg gcgacaaggt caaggaaggc tctgtggtcg tgatggtgga agctgctggt 240 gctgctgcag ctccggttgc agagccaaaa gcggcgccag cgcccgcagc atctgcgcca 300 gcagctcctg tttctgtagc agctccggtg gcagcgcctg cagcctccgg cccggtagag 360 gtgcgcgtgc ccgacattgg cgacttcaag gacgtggctg ttatcgaact gctggtcaag 420 gtcggcgaca ccgtgaaggt ggaacagagc ctgatcaccg tggagtctga caaggcctcc 480 atggagattc cttccagcac cgccggtgtg gtcaaggaaa tcaaggtcaa gctgggtgac 540 accgtgaaca tcggtgactt ggtcgttgtg ctcgaaggag tggctggcgc agcacctgcg 600 ccggcggcag ttcccgcagc tgcgctagct gcctcagcag caaccgctcc agtagctgct 660 gccgttgcag cacctgcagc tttggccgtg cctgcgcatt tgcccggtgg caccaccctc 720 gcccaagaga agaccggctc aaagaaggtc atcgccttca aaaacgccat catcgcggcc 780 ggtagccaag ctgtgcgcct gccctttatg cccaacgacc cgcgcgtggt ggactccacc 840 ggcgccttgg cgctcaagga agtgcccaag cgcatgttga ttttgggtgg cggcatcatc 900 ggcctggaga tgggcaccgt ctacagcacg ctgggcgccc gcctggacgt ggtggaaatg 960 atggacggcc tgatgcaagg tgccgaccgc gatctggtca agatctggca aaagatgaat 1020 gccaagcgct tcgacaacat catgctcaag accaagaccg tgtctgcacg cgccttgcct 1080 gaaggcatcg aagtgacgtt tgcgccggca gaggagggtg gcacggcccc cgcgccgcag 1140 gtgtatgact tggtgctgca agccgtgggc cgcacgccca acggcaagaa gatcagcgcc 1200 gagaaagccg gtgtggcggt gaccgaccgt ggcttcatca acgtcgacat ccaaatgcgc 1260 accaacgtgc cgcacatctt cgccatcggt gacatcgtgg gccagcccat gctggcgcac 1320 aaggcggtgc atgaagccca cgtggcagct gaagtgattg ccggtgaact gcaaggcaac 1380 aaagaattgg ccgctgctgc attcaacgcc cgcgtcattc ctagcgtggc ctacaccgac 1440 cccgaagtgg cgtgggtcgg cctcaccgaa gaccaggcca aggcccaagg catcaaggtc 1500 aagaagggct tgttcccctg gaccgcctcc ggccgcgcga ttgctaatgg ccgcgacgaa 1560 ggcgtcacca aactgctgtt tgatgactct ccggaagcgc acggccacgg caagatcttg 1620 ggcggcggca tggtcggcac tcacgcgggc gacatgatcg gtgaaatcgc actggctatt 1680 gaaatgggcg cagacgccgt ggatatcggc aagaccatcc acccgcaccc cacgctaggc 1740 gaaagcatcg gcatggcggc ggagattgcg cacggcagct gcacggatgt gccgccagcg 1800 aagaagtaa 1809 <210> 13 <211> 1400 <212> RNA <213> Hydra magnipapillata <400> 13 attcaaataa ccatgtcgaa gcataacata ttttaacgta gttttttgca agtcatcgtg 60 accatgtgaa tttgccagga gaaaaaaaaa gttaataagt taaagaaaga aataaacaaa 120 atcatcatga ctaagggttt ttacagttat ataaaagacc ctgacaaaat gaaacaaaat 180 ataacaactt tatacaagtt aggttcaact tcgtttgctg gagaataaaa ttaaagatgt 240 ctttatttga gaaagaagta caaatgaaag tttattatgt taaaaatcag cataatgaag 300 aaattcgtaa actttcagtg gaaattcgaa tggctgttaa ttatgaatac gttttggata 360 agatccgccg agaatttaca aatctgttag gaaaagatct tgacttgttt tataaagata 420 aagaaaatga tttcatttca atctcctctg atattgaatt gcagcaagcc tttaagagcg 480 ttgacaatag ttgccttaag ctttatgtaa aaataaagtc agctcattca aataaagaac 540 atgttggagt tacttgtgat ggatgcgatt ctaagatata tggaaatcgc ttcaaatgca 600 ctcaatgttt tgactttgat ttgtgctcat tatgctacaa aaaaggagag cacccatcag 660 accatgaaat gctgtcaatg ctgggaatta atgttgaatt ctttatggaa agttctcaag 720 taaaatctga tgaaactgag aatgaagaga gtttttgtaa aaagttaaat agcctaatag 780 aggttataaa tgagagattt ggagtagaca cagaactaat gcatactttg gttgacaatt 840 ttataaaaca gtataatcta aaatgcaatg aaagaaatag tggagcagac ataaataact 900 caaacaatga agtattcaga aataatggag gtgttaacca gactaatatg tcaaataaaa 960 atgatgtatc cattgataaa gacaaggatc ttgtcaagaa aaattgttta ggctcactgt 1020 gtcacaccaa tgttgaatca gtaccacatc ttgatttaaa caacgcttct gaagttataa 1080 aaaaacaaat aaatccactt ggttctttgt gtcctcaaac ctctgagttg caggttgata 1140 gttcacaaga ggggcttgca aattttattg gacacctata tcgtcatctt gtaactcaac 1200 agccaccaat aagtatagct aatgattttg aacttgttga taaagaaagt atggatcata 1260 aagaatcaaa gcttgaaaga tctcttaaac aaatggaagc gatgggtttt gacaatgaag 1320 gcgggtggtt gcgtcagttg ctaatttcta aagattgtaa cattgataaa gtgcttgatg 1380 ctttaagtcc tgccaaataa 1400 <210> 14 <211> 806 <212> RNA <213> Hydra magnipapillata <400> 14 gtttaagata tttttacgag aaaactgaca acttattttt tttttaggcg aagacgaaat 60 gttgcgttta tcactgcttt tgataatggt cacttctttg cttgcagatg aagattgcaa 120 tagggtatgt ttttctgtat atctaccagt ctgtggtagc gatggtaaca catatagcaa 180 tgaatgttta atgtcgatcg catcatgcat taaacgacaa acaattacaa aagttagcga 240 gggtgaatgt aatgacccag aagcaaactg taaaaaggta tgcccaaaaa tgtttcaacc 300 agtgtgtggg agtgatggta atacatacag tagtaaatgt gaattatcgg ttatgtcctg 360 caaaaaaaat tggactatta caaaactcta cgacggtgaa tgcaggcttc aagaactaaa 420 ttgtaatatg gcgtgtcctc aaataataga cccaatgtgt ggtagcgatg gccaaaccta 480 caacagtaaa tgtgaattat taatcgcagc atgcttaaaa caacaagcta ttattaaagt 540 ttatgatgga ggatgcaaca atgaaggaaa gtgtaatatt ccttgtaatc gaataaacgc 600 accagtgtgt ggtagtgatg gaaatatata ctcaaatgag tgtcttttga gaactgcttc 660 atgcaaacaa aagaaagcta ttacattcat acgaaatacc aatgacaaaa gttgttcatg 720 tctctttgaa tgcacaaaag aatataatcc tgtatgtgga tcaaacagaa taacgtattc 780 ttcagaatgt gtaatgcgtc ggtaca 806 <210> 15 <211> 737 <212> RNA <213> Hydra magnipapillata <400> 15 caactaaaaa gcaactatgg gtttatggct atcaaagtta gcatcagctt taaatgattt 60 tggtaataga agagctcgca ttcttttact tggtttagat gcagctggaa aaacaacaat 120 actttataag ttaaagctca acgaaaacgt cacaaccatt cctacaatcg ggtttaatgt 180 ggaagaggtg actccaataa aaaacgttac ttttaccatg tgggatgttg gaggtcagga 240 aaaaattcga cctttatgga gacactatta tcaaggttca gaaggactag tttttgttgt 300 tgatgcaagt gatgttttaa gaattcagga agctcgcgaa gagttatttt ctgtgttaaa 360 agatgaaggc atcagtaatg gaattcctgc agtgattctc gcaaacaaac aagatcttcc 420 taatgcgtta aaatcgtggg aattagttga taaaatgcgg ttaagagaat taagcggtaa 480 tccatggcat gttcaagaaa tgtgcgcttt aacaggggat ggtttgtatg aaggtattca 540 aaaacttgca gaaatggtaa aaacatatca aagagaaaac aaaattagat ggtgactgca 600 tgtagttagt ctacatgaat tttgtttctg taacaatggc gataaataaa aaaatatttt 660 gttaataaac ttttttataa aaatttttaa attgtaaata tattaactta tttaaatgtg 720 tatataaaat cttattt 737 <210> 16 <211> 357 <212> RNA <213> Hydra magnipapillata <400> 16 atgggaaaaa agaaggaatt atcagaagtt attagaggtc aaattcttat attagctcaa 60 caaaaaaatt caactagaga tattgcaaaa attgtaaatg tatcacaaac ttgtgttagt 120 aagacattaa gtagaattcg acaatgtgga acattttcgt cacatgggag aagtggcaga 180 ccaaaaaaag tgactgaaag gttgacaaga cgcatcaagt ttattgtgga gaataatccc 240 aagctgtcat catatgggat tctagatgat ttgagtcctg gaaatgctga agcaagtcta 300 ccaagcacaa gaacgatcag acgtgtgctg cagattgatc tgaagcatcc tgcttga 357 <210> 17 <211> 749 <212> RNA <213> Hydra magnipapillata <400> 17 agagagagta tgctactgta tataagggag ttagcagatc acaccatata atggttgcaa 60 tgaaggaaat acgtttacaa gaagaagaag gaattccatt tacagcgatt agagaagcat 120 cattgttaaa agagcttaaa cacgctaata ttgtgacctt gcatgacatt ataccaacaa 180 aggagcatct tgtactagta tttgaatact tgcacactga tctaagcata tatttagaag 240 aaagaccatt tggacttcac cctactaatg ttaagctttt actatttcaa cttctgagag 300 gtgttcgata tattcacaag aaaaaaattc ttcatagaga cttaaaacca caaaatgtac 360 ttcttaataa aaaaggagag ttaaagttgg ctgactttgg cttagctcgt gcaaaatctg 420 tacctagcca tacttacaca aatgaaattg taacactctg gtatcgtcct ccagatgttt 480 tgttagggtc tacagaatac agtacctcgc tagacatgtg gggtgttggc tgcatatttg 540 tggaattggt atgtggtatt ccattgtttc ctggtgtgaa aagtccacat gatcagttga 600 caaaaatttt ccggttgcag ccagaaaata gaatatcagc tgaagaagcg atgaaccatg 660 agtattttaa ggatttacca gaagaaattt ttactctcgt agattgttta ttccgagctg 720 gtacaaactc agaaaaaagt tacgagtaa 749 <210> 18 <211> 1692 <212> RNA <213> Hydra magnipapillata <400> 18 atgtatccat cagattgcgt tccacttaga atctacggaa tggttaaagc tcacatacca 60 gaaaaaaact acctaatgcg tcctgttatt tccattatta acacaccacc ttatgaaaca 120 tcagagtatc tgggtttaag attgcaaaat ttacaaaata atgttcatta tacttgttct 180 ccgtgcggta agcataactg ttttgtgtgt ttaactaaat gcgaggcttg cagaaactta 240 aattgttcat gcagaacaat agagtatgga aatgaaattt cgagagtgaa accaaagcct 300 agtatctaca ataattcact tattaaagtt gggggagttg gagatttttg cgaaataaag 360 tcttttagag atgctaactt accagaagta ctaattgaaa accttagaaa gcttttatac 420 aaggttccta cacctgttca gtcgatagca ataccaatat tactagaagg caaagattta 480 gtagctcgtg cagaaactgg atcaggaaaa acggctgcat ttttaatacc aattgtttca 540 accttaatta aaagaaaaac agtgaattca atgaatccat ttgctgtaat tttaactccc 600 acacgagaat tggctttaca aattgagaga gaagctttgt tgttgacaga aaataccttt 660 ataaagccag tatgtaccta tggtggaata actattggtc atcagaagaa aaaaattgca 720 aaaggttgtg atatacttat tggaaccctt ggaagaatcc ttcatcttgc taaacaatca 780 atgctctcct taaaagaact caaaatactt attattgatg agggagatca tatgctagat 840 aaaaattttg ttaaagatat tggaaaatta gtgcatgact ttgatatgcc tgaaaaatct 900 ctacgacaga ctgctttatt ttgtgcaaca ataccgacta ctcaaataat ccatgatgac 960 aaaggagtta aaaaaaaatt agcatttttt actgatactg tttgcatgtt tttaaatgat 1020 acatactgtc atatagatgt tggtaacaat ttggttacac caaaatccat aattcaacaa 1080 tttttgtttg tagataaact ggaattaaaa tttcaattat tagaaaaaaa aatcgctgaa 1140 gaactatcaa atgaaaataa aactattgtt ttcattgaaa ccattaaaga aacaataaaa 1200 gtagctcaac agctgtgcat taaagattac cctgtaacag ttattcatgg tcggcgtact 1260 caaaatgatc ggaaacaagc tatgtatgaa ttcaatgaag gtttaaaacc tcttttgata 1320 tgtaccaatg ttagtgcacg tggtcttgat atttttgaag ttaaaacagt aataaattat 1380 aatttaccga atcttactca tgatgatatt agaacatacg tatatcgaat agggcgtgca 1440 ggtcgtattg ggaatccagg caaatcaatc tcattgtttt tgaaaggaag ggatgaagtc 1500 ctagctaaac cattggttgt tttattagga gggtctcaac aagaggtacc aacatggcta 1560 gaggagcttt gcttagttcg tgaaatggca aaattgggta actattctca agcaatttat 1620 gaaacaaaaa gggttgcaat gcttacagag aaaaattcta gtgatgatac taggaggact 1680 attttctgtt aa 1692 <210> 19 <211> 2174 <212> RNA <213> Hydra magnipapillata <400> 19 atccatagta acgttacaaa taaatgaaaa tgatttttaa aatgaagaag cagcttttgt 60 catctttttt taatatcact gttaacatta tatttgttgg cggcctaatt ttatcaaaaa 120 gttcaattga gttctgtgaa aatacaggat caagcaatga tctaaaggat cccacaaatg 180 tagtaaccca atcagcatgt gaaaaaaaga tggttgtttt gttgtctgtt ggaaataagc 240 agggagaaac tgaaaagctt caggctgttg tttctgttgt tcaaaactca gccacaaatg 300 aatttgcaag actatataat ccatttatga taactgtttc aaaatcacct gtttatctta 360 acttcccatt cttttttaat gggataacag taaataatca accttatgaa gaaataattt 420 tgagcaaaaa tcattggtat gtttcagata gttctcgtca atgtttagac caatggcaag 480 tggaagaaga agatgacgaa catccaactt gtggctacca atatacaaac agcactcaaa 540 aacaaactga tggaacatgg aagactgtca aaacaagaat ttgggacagc cagggatttt 600 gttgctattg tacgcaggat ttaaaaaact attatattaa aaaagatatt caagatgcta 660 acagagctgg tattatttgc aaacctttga caaattcccc tcaagcatct gctcattgta 720 tgagaatgag caacttatgg tacactttaa atgagtttac tgagtcctac agagattttt 780 cgatttatgt gaaagctttt gaccaaatta caaaagttgt acaaaataaa tcatacatag 840 attatgttaa tggtggtgag atacttttat caccaagtca aaaaagtgca actggatctt 900 acaacaggat aacagggaat tatgttggtg atcttcaacc tattaaaagc tatcctgtat 960 taacaaacaa ctacttttta attccattca gctctacaaa tgttgatccc aaaaaagaac 1020 ctcagttaaa gagtggaatt tcaaagtgga tgataatacc tcgcgattta gtcagtacag 1080 atgcaaagca atgcgatatg attggtgttg gttattcagc attcagaaat caagctgctt 1140 atggaacggg ttatggatgc agagccaaaa aaggatcatg tttggctaat caaccttata 1200 acaaatttat ggatgatgag gatcgattag aaaaaggtaa aatgccttgg tattttccag 1260 caaggtatgg aaaattagca ggtgttaaac aaaatattgg cgacaatgat aaataccttt 1320 taacatatga gcttgatgat gaacaaatta gcctggttac attacaaatt agtgctgatg 1380 atgttgtatt agtctataac agagctaccg gtataataac acgcactgct attcaagatt 1440 ttgaagctct ctcattggaa ggtcaactta gtgttgatgt tttgaatact ggttatgttt 1500 catctgattt tagaatttca atcccatcat gcacatctgg tgttcagcca atagaagaaa 1560 agcgtatcac tattgatcct cagatgacag agactattac attcaaaatg atgacttcca 1620 cagataaaaa aagcgcacat gattgcacaa ttaatttata tgactcaaaa aacatactgc 1680 tacaaagtcg caattttaca ttttctacaa aagcaccatg tgtttgtgaa gtacaatcat 1740 gcaaatgcga ttgctccgaa ggtggtggcg taaaatgtgt gcaagctgaa ggaaagttta 1800 tagataatcc aaatctcttt gtaccacaat ccacaggttt tttggacaag ctgtggagta 1860 gtataaaatc ctttccgtct attataggta acttttttag tggaatattt ggctctttat 1920 tcggagatct ggttcagtat gcaatatttg cagcaatagc actggtagta atatgtttgt 1980 gttgcaattg tggtggtttt agattgctca agagattcat tcctaaattc aataaaaaag 2040 ggtataaaca tcttaaacta aaaagatacc aagaacaaat atataagcca aaagagatta 2100 ctaatggaaa tccaactaca gccatgcaaa gtattacacc cgtacctagt atgcctatac 2160 cgtcaaatac ctaa 2174 <210> 20 <211> 1660 <212> RNA <213> Hydra magnipapillata <400> 20 gatcaattac taacattgca acgattagca ttagtaaaag tttatttcta aaagttatct 60 tttttgcata atcatcgatt aaatattact gtataaatgg cgttatcttt atgtaaaacc 120 aaagacttgt tttcaagtcg cgaaggctta aatctcgata aattttcatt taattacaaa 180 caagacagtc aattttccgt atttcgtgat tatcttggac ttataagact gatactacct 240 gaaaatcgca tagaaatgtc taaattgcat tcgcctacag aatctgtgga taatgttgat 300 ttttttttta attatccaaa tattatatcc tcaaacaggc atcgaattga tagtttagat 360 tcagatgaga cgagtgagag ttcttctagc gaaggtcgag gtgataatga agtaatatat 420 gaaaacctaa gctcttctga agagtactct ccctaccggc aaagtaaagt caattcaatt 480 catagtaatg ttttggcaca aacatacgca caaacattag agtctcttat tgctaaccaa 540 agtgtagtta gtcaacaaat acataatcat gttcaacaac aaattcaaag caaagctttg 600 aaaaatctta gcaagacatc ggtttgtgtt ttttgtcgaa ataatggtga gtccagagaa 660 ttttacagct cgcacacttt aaaagataac gaaggaaata ctatgtgccc aatacttaga 720 gcctacacat gcccactctg caagtcacat ggtaaccaat cacatacaat taaatattgt 780 cctaaatata ctccaaaacc gaaaacagat aaattattag atatcagcat gcctttactt 840 tagcttacga tgaattttta tttgatattt aaaaatgaaa gaaatttaat tttgtttagt 900 gaggtataac tattttttat gtacatatct aagttctctt gcgcgcattc ttgctgcgca 960 agattgtttt gttttgcatg ctctattttt ttaggtgagc ttttaaattt ttacgttcat 1020 tttttcattg atagttttaa attaatcctt caaaatattt tcattattat cctagttaaa 1080 ttttaaaggc agttttttaa attatacagt ctcttgattt tatcttattt tgtttattcg 1140 tcttatttaa tgtttgtata taacgaccgc agaaaatgct tagcgttcca tagtttattt 1200 aaaattcttt gtctcatctt acagtatgat aatatttttc tttatttgaa caaccttcat 1260 catataagct tttcgtcgta ggctttttta aatttaaaaa ccgccaatta ctggtatctg 1320 taaaaaaaaa aaaactacag gtgttacatt aaatctgttt actaagtaat atttaatcta 1380 tgtgaatgtt cctttatgtt ttaatatttt taacaatgta atcaagtagg actaaaactt 1440 tcttattttt ttcttttcaa ttttagtcct aattttagat aaaatttaat cgcgattatc 1500 attttatgat ttgtacatgc aagaattttg tggtgttaag aaaaccttca tatttgtgtt 1560 tatatattta aacgctgtaa atagcgtgtc aaagattctg tattagattt tacgccatat 1620 tgtatttagt ttttattcga atataacgaa tgcagatttt 1660 <210> 21 <211> 748 <212> RNA <213> Hydra magnipapillata <400> 21 catatagcgt gtttatgttt tttgttttcg tgacaacaca caatttaaat taaacgttat 60 gtcgtcgtta atgtcatctc aaggcccgtt gaaggccatt catattcaag ttccaattgg 120 aattccaact ccagatggcg gattagaaat tcctaatcgt gttttcttgg gaggcatacc 180 caccgagaca actgaattag agctggagtt gtttttttct gattacggat tagttaaaga 240 tgtgcgaatt gtaacagacc gagttactgg tgaatgtaaa ggttatggat ttgttacatt 300 tgatgaaaat gaagatataa acgagcttgt tgttaagaaa tccatcaata tgaaagggaa 360 aaagcttcgg gttagaaaag cgattcgacg caacggttcc caatttaaca gttgtggtaa 420 ttcaagtgaa tgttctccaa gtggtggcaa ctctccgctt tctaaaggtt ttgatgaaaa 480 cgatcatcaa tttttgttaa ttcctcaaca atccagtact ccttctcagt ttcctatttt 540 gtcatcacat atgatgtatc ctacaagttc tcatatatat acttcagcgg gtcctatgac 600 agtaacgcct cgcattgtac cgtcgcctca tttaattgct ggaacgcagc cataccatac 660 tcctgttatg tatcctgtgt actactttcc ttgtgccaat cagcaaatgc aaatgaactg 720 ttcaatgatt caacagatga caatttag 748 <210> 22 <211> 1573 <212> RNA <213> Hydra magnipapillata <400> 22 taaaataata agaaaaaaat taaattttga gttttgttat ttttctgttg tttttttgtc 60 tttgtctacg gtcaaaattt acaaaatgac aaatgaagct agtgaaatat ctaaaaacga 120 gttttttaat gaaataaagt ttggtttaat gaactctgaa ggcttttatc taacttcgga 180 gaagtttgga aatcaaatta gtgtcactgg aacttcgttg cgcagtaagc aagtttggca 240 ctttgaaaaa agtaaatcct cgtcgatgaa aggttactta agaagtcctc atttaaagta 300 tttagagtca gacaaaaatg gcgtcgttac ttgcaatagc gatactaaat gtgattcttt 360 tctatttgaa gttgaactta cagacgaagg taactggatg tttaaggatt gtagtggaaa 420 atatctttct ggaactagta aaaatattaa ttgccaatta aaagaaaaag cggggctaaa 480 tgcaatatgg gcgatccata ttacaaatca tcctcagtgt aatataatga gtgtagctcg 540 gaaaagatat gttcacgttt cagatgaaga atttagagct gacgaagatc ttccgtgggg 600 taaagaatat gttattacaa tcgaattttg taatggaaag tatgctttta gagatttaag 660 cggtcgatac ttaaatggtg taacaggtta tttagatgaa aaatgtagca atgacacact 720 ttttgtactt tatctaaacg gattaatgta cggctttaaa tgcaacaata ataaattttt 780 aactgttcaa ggtagcaagg gaaaactaat tgcaaataaa gataactttg gcaaagatga 840 acagtttttg atcgaagaaa gtaaacctca atgcattttt actgcaagca atggcaaaaa 900 gttctctgta aaacaagggg ttgatgttac tgccaatgtt tttgaagagg ctggtgcgtc 960 tgaaatattt caaattggtt ttgacataaa tagagaggat tgcgtaacca tagttacaaa 1020 tttaaaaaca tacctttgta caacagataa atcagtcatt gccaagtctg atatatctat 1080 gaacagttat tttcaaatgg agtggcacga tcaacacgtt atgctaaaaa acttttgtgg 1140 aagctatata acttcatcgt ccggtgggaa gctttcactt gtatctgaga ataacaaaga 1200 tgaaaactct ttatttacaa tccaaatagt caatcgccca atacttgttc ttcgttgtga 1260 atatggttac gttggattat catcatcatc aacaaaagtt atgtgtaacc gtggtgctgg 1320 aactgcaatg tacgtatcaa acgaaagtgg aaagtatcgt tttaagtttt ccgacggaaa 1380 gtcatggaaa ataaaagatt cagattcaat gattttttct gatttagaag gtgatttgtt 1440 cttttgccag cttcatagta aaaacagaat ggtaattctt gctccgaatg gaaaatattt 1500 aagaagtgat aaaaatggaa atatttacgc tacagaggag gaagtgaagc catcaattta 1560 ttgggagtat taa 1573 <210> 23 <211> 1503 <212> RNA <213> Hydra magnipapillata <400> 23 atggcagtaa atgctcctct tgacctaaaa tcctatcttc gatatgttga cgatagtcat 60 gctagatttt ctaacgctca agaagcagaa caattcctaa ttatcttgaa caaacaacac 120 cctccaatac aatatacaat cgaaattgaa agtgaaaata gaactctaaa cttcctcgat 180 ttaaccatag taaataacac caaaggtaaa tacgagttta aagtttacag aaagaacgcc 240 attacaaata ttcaaattaa acctcactca aatcatgacc ccaaaattct aaacgcaata 300 tttaaaggtt acgttcacag agcttactct atatgcagtg atttatattt agaagatgaa 360 attaatttta tatttcacct tataaacccc gttactttac aattagaaaa ctttattact 420 caaagtgata aaaaaaatga atggaactca atattgaaag ttaaaaatgg agaaaaaaaa 480 acttgcgtaa ttgattgtgg agtttgtgga gataaaagtt ctggaaaaca ttacggagtt 540 aacacttgtg aaggttgcaa aagttttttc aagcgaagcg ttagaagaaa cttacaatac 600 acctgcagag caaaaagaaa ctgttcaata gatcaacacc atcgcaatca atgtcagcat 660 tgtcggctta aaaaatgtct aaaggctgga atgcgtaaag atgctgtaca gcgtggcagg 720 ctaaatagtc aacaaggagc tgcacaggtt tttgaagatg caacagttaa taacttttca 780 tttttatctg ggtttgttac tttattacta agagccgaac cttgtccaat atttcgttat 840 tcacaaggag tgtcaaacaa cccccaattt gattttatcg atatcgacaa ctatgagtta 900 gcagctcgcc ttctttttaa tgctgttgaa tggtctcgca acataccgtt tttcccaaat 960 ctttcattaa ccgatcaaat agccttactt cggttatgtt ggaaagagtt gtttattctt 1020 aacgtggcgc aatgtccaat gctaatagat gtatctcact tgttaaactc tcaaatgaat 1080 atttatgcat ctcctgagca catggcatct tttttagacc aagtccggat attaaaagaa 1140 caacttaata aactgagggc aatgcacgtg gatccagctg aattcgcatg tttaaaagca 1200 atagttgtat tttcttcaga tgcacccggt ttgaacgatc ctcaatacat agaaacttta 1260 caagagaaaa cttattttgc acttgaagac tatataaaaa ctcaataccc cttgcaacca 1320 actcgttttg gaaagttatt gcttcgattt ccctcaatac gaataataag cgcaacagtg 1380 attgagcaac tattttttgt gagattggtt ggaaaaactc caattgaaac attaatacga 1440 gatattctta tttctggtac atcatatgat tggcccactt taaactactc tgaaagaact 1500 taa 1503 <210> 24 <211> 2643 <212> RNA <213> Hydra magnipapillata <400> 24 atgacccgcg acatcgagcg gggcacccgt ggcgtgcatt cgctgatcag ctattcgctc 60 tacagcatcg ttcccaccct gatcgaggtg gcgttggtgc tgacgctgct ggcggtcaag 120 ttcgatgtct ggttcgcctg gatcaccggc attgcactgg tggtgtacat cgccttcacg 180 gttacggtca ccgagtggcg cacgcagttc cgcaaaaaga tgaacgagat ggacagcaca 240 gcccacagcc gcgccatcga ttcgctgctg aactacgaga cgctcatcat cgccagtgca 300 ctggtcacca tgttgtggcg tgctacccaa ggcgtggtgg acgggcgcat gacgctgggg 360 gacctggtca tggtcaacgc cttcatgatc cagctctaca ttccgctaaa ttttctgggt 420 gtgatctacc gggagatcaa gcaaagcctg accgacttgg agaaaatgtt tagcctgatg 480 gagcgtgagc gcgaaattgc cgatgcaccc gaggctcccc ccttgcagtt ggcggccgat 540 ggttcggacg ccagcgtgcg gtttgaaggt gtcactttca gctacgaccc cgcaggcagc 600 aaggcagccg caggcaccga agcgcgcacc atcctgcacc acatcagctt cgagattcct 660 gcgggcaaaa ccgtcgctgt ggtggggccc agtggttcag gcaaatcgac gctggcgcgc 720 ttgctcttcc gtttttacga tgtgcagcag ggccgcatca ccattgccgg gcaggacatc 780 cgcaatgtga cccaaagcag cgtgcgtcag gccatcggca tcgtgccgca ggacacagtg 840 ctttttaacg acacggtgga atacaacatc gcctacgggc gacccggtgc cagccgcgcc 900 gaggtcgagg cagcggccaa agcggcgcac attcacagct ttattgcggc agccccccgg 960 ggctacgaca ccatggtggg tgagcgaggt ctcaagctca gcggtggaga aaaacagcgt 1020 gtggcgattg cccggacctt gctcaagaac cctcctgtca tgatctttga cgaagctacc 1080 agcgcgctgg acagtgctaa tgagcgcgcc attcaggcgg agctgcaagg cgtttcccga 1140 aacaagacaa cgctggtgat tgcgcaccgc ttgtccacgg tcgtggacgc tcacgagata 1200 ctggtgatgg atgcaggtca catcattgaa cgcggcaacc atgccgaact attggcggcc 1260 aatgggcgtt acgcgtccat gtgggccttg cagcaaagcg gggatctcct tgccgtccga 1320 caaaatggga agcgcttcgc cgaactggcc ggtcgtgact ttcggtttgg tttgctcgat 1380 tccttcatgg gagtgcgagc gcaaagtggc attccccaag ttgcccaaaa cttccaggaa 1440 atactggttg tcagttgcgg cggcaatctc agcgtggaac cggtgccgcc agcagcggta 1500 tcaaaggtta aagaggagcc tcttgtggcg caattgcaat taaaaaaaat cgagaagaca 1560 taccccaacg ggttcaaagc agtgcacggc gtggatctgc aaatccgcga cggtgagttc 1620 atggtgtttg tggggccctc gggctgcgct aaaagtacca tcctgcggat gattgcgggg 1680 ctggagagca tccgtggcgg tgagctgttg atcggtaacc aagtggtaaa cagcttgccg 1740 cccaagcaac gcggcattgc gatggtgttc cagaactacg ccctgtaccc gcacatgaag 1800 gtctatgaca acctggcgtt tggtctgaag ctcgcaggaa ccgcaaaagc agaaatagat 1860 gcccgtgtgc gggaagctgc caagctcttg gaaatggacc atctgctgga ccgcttcccc 1920 aaacaattgt ccgggggaca ggcgcagcgg gtggccgtgg ggcgcgcgat tgtgaaacgc 1980 ccggaagtgt tcttgttcga tgagccgctg tcgaatctgg atgcgaagct gcgcgcctcc 2040 atgcgcgtac gtttgaccga attgcatcgc accttgcggg aacaagggcg tccatctacc 2100 gtggtgtacg ttactcacga ccaggtggaa gccatgacca tgggggagcg gatctgtgtg 2160 ctgaaggacg gtgaaatcca gcaagtggat acccctacgg ccttgtacga caaacccgcg 2220 aacgtgtttg tggcgggctt cattggttca ccggagatga acatcatccc tgcagaagtc 2280 acccagaatg gcgcgcaagt ctccattggc ggtgtcacgc tccaagtccc gcaaaagcac 2340 attccccgcc tccaagcact aaaggcagtc aagtttggta tccgtcctga gcacatcacg 2400 gcgggtggcc atagcgaatc cgctgtgcag atggtggacg gcaccctgcg ctttatggaa 2460 cacatgggca gcgaggtgta cgtgcacttc accctgggcg atacaccgct gactgcccgt 2520 gttcccgcag accaactccg cgatcttgca ggcaaagccc gcggggatcg ccatgccttc 2580 ggtatccaga tggatgcctg ccacgctttc gacatggaca gcgggctgaa tttgttcttg 2640 tga 2643 <210> 25 <211> 1338 <212> RNA <213> Hydra magnipapillata <400> 25 atgtccaaaa aagtctttat caaaaccttc ggctgccaga tgaacgagta cgactcggac 60 aagatggctg acgtgctcgg cgcagcgcag ggctacgagc ccacagacga tgtggaacag 120 gccgacctga ttctgttcaa cacctgctcg gtgcgcgaga aggcacaaga gaaggtgttc 180 agcgacctgg gccgcatcaa gcacctcaag gccaaagggg tgcaaatcgg ggtgggcggc 240 tgcgtggcca gccaggaagg cgccgaaatc atcaagcgcg caccttatgt ggacgtggtg 300 ttcggcccgc aaaccctgca ccgcctgccc gaaatgctga atgcccgcaa agcgctggac 360 aagccgcagg tggacatcag cttcccggag atcgaaaagt ttgaccacct gccccccgcc 420 aaggtggacg gtgccagtgc cttcgtgagc atcatggaag gctgctccaa atactgcagc 480 tactgcgtgg tgccctacac ccgcggcacg gaaatcaacc gtccctttga agacgtgctg 540 gtcgagattg cgggcctcgc cgaccagggc gtgaaagagg tgaacctgct gggccagaac 600 gtgaatgcct ggcgcaacgc catgggcgac tcgggcgaga tggccgactt tgccaccttg 660 ctggaatatg tgagcgacat ccccggcata gagcgcatcc gctacaccac cagccacccc 720 aacgagttca cgcccagcct gatcgcggct tacgaaaagc tgcccaaact ggtgagccac 780 ttgcatttgc cggtgcagca cggcagcgac aaaattttga tggctatgaa gcgcggctac 840 accgccatgg aatacaaaag cactatccgc aagctgcgcg ccatccggcc cgacatgagc 900 atcagctcgg acttcatcgt cggcttccct ggcgagacgg atgaagacca cgcgaagatg 960 atgaagctga tccatgacat cggctttgac aactccttca gcttcatctt cagcccccgc 1020 cccggcacgc cggcggccaa cctgcacgat gacaccccac acgaagtgaa gctggcccgc 1080 ctgcaagagc tgcaagccgc catcaacgcc aacattgcca ctatcagcaa ccaacgcctg 1140 ggcacggtgc aacgcattct ggtggaaggc ggcagcaagc gggacaacgg cgagctaatg 1200 ggccgcaccg agtgcaaccg ggtggtgaac tttgctggta atccacgcct ggtggggcag 1260 ttggtggatg tgacgatcac tgagacgcgc agctacacct tgcgcggtga ggtgctgact 1320 gcagagcacg caacctga 1338

Claims (11)

A microarray chip for detecting whether acetaminophen has been exposed, in which all of the oligonucleotides of each nucleic acid sequence of SEQ ID NOs: 1 to 25 or the complementary strand molecules thereof are integrated.
According to claim 1, The gene is characterized in that derived from Hydra ( Hydra magnipapillata ), microarray chip for detecting the presence of acetaminophen.
1) separating RNA from the hydra of the experimental group exposed to the test sample and the hydra of the normal control, respectively;
2) synthesizing cRNA labeled with different fluorescent substances from the RNA of the experimental group and the control of step 1);
3) hybridizing the cRNA labeled with the different fluorescent materials of step 2) with the microarray chip of claim 1;
4) analyzing the reacted microarray chip; And
5) In the analyzed data, a method for detecting whether or not acetaminophen is exposed in a sample, comprising the step of confirming the degree of gene expression integrated in the microarray chip of claim 1 in comparison with a control.
[4] The method of claim 3, wherein the sample is selected from the group consisting of biological samples, food samples, chemical samples, industrial samples, clinical samples, and environmental samples.
According to claim 3, wherein the fluorescent material of step 2) is Cy3, Cy5, FITC (poly L-lysine-fluorescein isothiocyanate), RITC (rhodamine-B-isothiocyanate) and rhodamine (rhodamine) selected from the group consisting of Characterized in that one, acetaminophen exposure method.
1) separating RNA from the hydra of the experimental group exposed to the test sample and the hydra of the normal control, respectively;
2) Quantitative real-time reverse transcript polymerase chain reaction using the RNA of step 1) using both primer pairs complementary to each gene of SEQ ID NOs: 1 to 25 and capable of amplifying the gene. , qRT-PCR); And
3) comparing the gene product of step 2) with a control to confirm the expression level, a method for detecting whether acetaminophen is exposed in a sample.
The method of claim 6, wherein the sample is any one selected from the group consisting of biological samples, food samples, chemical samples, industrial samples, clinical samples, and environmental samples.
A kit for detecting the presence of acetaminophen, comprising the microarray chip of claim 1.
The kit according to claim 8, wherein the kit is any one selected from a group of fluorescent materials consisting of streptavidin-like phosphatease conjugate, chemiflurorensce, and chemiluminescent. Characterized in that it further comprises, acetaminophen exposure detection kit.
The kit according to claim 8, wherein the kit comprises a buffer solution used for hybridization, reverse transcriptase for synthesizing complementary DNA (cDNA) from RNA, deoxynucleotide triphosphates (dNTPs) and ribonucleotide triPhosphates (rNTPs), pre-mixed or separated supply type, labeling. Kit for detecting the presence of acetaminophen, characterized in that it further comprises any one selected from the group of reagents and reaction reagents consisting of a washing buffer solution.
A kit for detecting whether acetaminophen is exposed, including all primer pairs complementary to each gene described in SEQ ID NOs: 1 to 25 and capable of amplifying the gene.